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Cheers and Jeers - Oct. 17, 2014
Thomas Drance
October 17 2014 03:06PM
It's Friday, the Canucks haven't played in forever, and now it's time for: "Cheers and Jeers!"
This regular feature was originally started by Canucks Army forefather Cam Davie during the 2011-12 hockey season. Now that I'm the old hand at this here Canucks hockey blog (that happened... quickly), I figure it's my turn to carry on tradition.
This week, on the other side of the jump, I give a high-five to Willie Desjardins and Bo Horvat and wag my finger at Milan Lucic and beat up the dead horse that is the Canucks' recent draft record...
Comets Militia: Down on the Farm - October 17th, 2014
Josh W
October 17 2014 09:30AM
Courtesy of UticaComets.com
This past weekend, the American Hockey League finally started up its regular season with the Utica Comets playing their first two games. It's looking to be a very exciting team this year with plenty of firepower, some good defence and two solid goaltenders in net. The depth is looking so good that the Comets had to send a few popular Canucks prospects down to the ECHL to give them more playing time.
With that being said, read past the jump to see how this week has gone for the Comets.
Nation World HQ
October 17 2014 08:00AM
Jonas Hiller seeing beach balls, the Oilers aren't very good at hockey, the Leafs could use Eric Staal, the impact of defense on goaltender stats, loving the game again and more in this weeks Roundup.
Trade Targets for Defensive Depth
J.D. Burke
October 16 2014 05:00PM
Should the Canucks bring B.C. native and VANCOUVER GIANTS LEGEND Cody Franson home?
If there is anything we can glean from these first two games, it's that the Vancouver Canucks are short on defensive depth. As much as the change around town is relished and providing solid early returns, this is an area of concern all the same.
This franchise could sit on its heels and watch the wheels spin without so much as batting an eye, but they've every intention of making the playoffs this season and standing pat would bode poorly for their chances. Injuries are a nasty occurrence, and they've always a knack for striking Canucks defencemen at the worst of moments.
Before you know it one injury to the blue line has turned to three and having a good sixth or seventh defenceman won't be enough. Yann Sauve should never enter an NHL arena as anything more than a fan, let alone the three games he did last season as a Canuck.
Lets look at one of the Canucks biggest issues with their roster and what they can do about fixing it.
Adjusted Save Percentage: Measuring the Impact Defense has on Goaltending Statistics
money puck
October 16 2014 01:30PM
One of the reasons we love sports as much as we do is because of the great debates it can spawn. I came across a classic hockey debate the other day in relation to Ryan Miller. "Of course Miller didn't post elite numbers last year. He played for Buffalo!"
As I was thinking about this, it struck me that while we have made huge strides in advanced statistics overall, we haven't made much progress in terms of quantifying the impact of defense and separating that contribution to statistics for the goalie themselves. We can tell the percentage of shots a goalie save (SV%), but we have little to no insight into the difficulty level of the shots themselves.
From a quantitative standpoint, we understand the game better now than ever before. We understand shot attempt differential, which we know we can be used to approximate possession. We call this corsi, and we understand this to be a key component of overall team success. We can also begin to isolate the impact a player has on their team's shot attempt differential, which is a solid representation of strong two way play.
However, there is one factor that detractors of advanced stats have always struggled with. Detractors have long protested that not all shots have an equal chance of going in, and they're right. Is it possible to adjust save-percentage to reflect easier or more difficult workload? Find out after the jump.


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Simple Date Rolls
|Canned water chestnuts
|5 Ounce
|Pitted dates
|Bottled french dressing
|1⁄4 Cup (4 tbs)
|Curry powder
|1⁄2 Teaspoon
|Bacon strip
|10 , cut in halves
Drain water chestnuts and cut in halves (you should have about 20 pieces).
Stuff dates with the pieces and put in a small bowl.
Mix French dressing and curry powder and pour over.
Let stand several hours, stirring often.
Drain dates and wrap each with hal
Rate It
Nutrition Rank
Nutrition Facts
Serving size: Complete recipe
Calories 1117 Calories from Fat 531
% Daily Value*
Total Fat 60 g91.6%
Saturated Fat 15.6 g78%
Trans Fat 0 g
Cholesterol 29.5 mg9.8%
Sodium 1878.6 mg78.3%
Total Carbohydrates 128 g42.7%
Dietary Fiber 13.3 g53.1%
Sugars 99.1 g
Protein 17 g34.6%
Vitamin A 6.8% Vitamin C 7.4%
Calcium 8.2% Iron 21.9%
*Based on a 2000 Calorie diet

subset: commoncrawl


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Editorial Contributor
This project received 4 bids from talented freelancers with an average bid price of $78 USD.Get free quotes for a project like this
Project BudgetN/A
Total Bids4
Project Description
A lifestyle magazine (in print and online) dated to launch in September seeks critical thinkers and writers from around the United States who can contribute to its assemblage of stories, news, reviews, and editorials—developed around the experiences and mental constructs of gay Asian men.
We seek to explore the culture blossoming around this community especially in the fields of arts and entertainment, culture, politics, fashion, and food. Contributors are compensated per assignment.
If this opportunity interests you, please send a resume, optional cover letter, and work samples to danielcontributors at gmail
As a project emerging from combined passions for dialogue, storytelling, and appreciation for the creative and intellectual forces of the culture upon which we focus—we intend to value and work closely alongside those who join us in spearheading this movement.
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- Forbes
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- Wall Street Journal
- Times Online


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How to Give Your Bike a Tune-Up
Spring is here! And that means it's time to dust off the bike and start pedaling. Here are a few tips on how to give your bike a maintenance check before you ride.
Maintain proper tire pressure
Always inflate bicycle tires to the recommended air pressure (as noted on the tire's sidewall). Some tires can handle an inflation range. When inflating these types of tires consider the rider's weight, as well as any loads that may be carried. Higher pressure gives better performance on pavement and other hard surfaces, while lower pressure works better for off-road rides. Gas station air hoses may inflate a bicycle tire too rapidly and often give inaccurate tire-pressure readings, which can result in a blowout. Whenever possible, use a manual hand- or foot-operated pump with an accurate gauge. It's also a good idea to pack a good air pressure gauge whenever you go out for a ride; choose one that can read up to 120 psi (pounds per square inch).
Check your brakes
It's a good idea to regularly inspect your brakes before you go for a ride. Squeeze each brake lever toward the handlebar to make sure the break moves freely and stops the bike. If the break lever reaches the handlebar, it needs to be tightened. Now, check out the brake pads: They should be about 1/16 to 1/8 inches away from the rim when the brakes are not applied — if you can stick a matchbook cover between the rim and brake, your brakes are not properly adjusted.
Preserve your bike chain
Want to extend the life of your bicycle chain? Lay off the oil. Yep, you heard that right. Machine oil does a fine job lubricating bike chains, but it also attracts dirt and debris that could wind up damaging them. Instead, use a dry chain lubricant. It will keep your chain running smoothly without sucking up dirt. You can buy dry lubricants at most bike shops.
Thanks! You're on our list.
And about to get a whole lot healthier.
Some people like to travel by train because it combines the slowness of a car with the cramped public exposure of an airplane.
Dennis Miller
I think my pilot was a little inexperienced. We were sitting on the runway, and he said, "OK, folks, we're gonna be taking off in a just few—whoa! Here we go."
Kevin Nealon
"I can't wait until your vacation is over." —Everyone following you on Instagram
A man knocked on my door and asked for a donation toward the local swimming pool. So I gave him a glass of water.
Comedian Greg Davies
Just found the worst page in the entire dictionary. What I saw was disgraceful, disgusting, dishonest, and disingenuous.
Client: We need you to log in to the YouTube and make all our company videos viral.
From clientsfromhell.net
My cat just walked up to the paper shredder and said, "Teach me everything you know."
"Just because you can't dance doesn't mean you shouldn't dance." —Alcohol
@yoyoha (Josh Hara)
My parents didn't want to move to Florida, but they turned 60 and that's the law.
—Jerry Seinfeld
Q: What do you call an Amish guy with his hand in a horse's mouth?
A: A mechanic.


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Developer Essentials for FileMaker 11 Exam
FM0-305 Questions & Answers 115 Questions
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Let us know when your receive great advice so we can thank our lawyers and point others with similar questions in the right direction.Got it!
It's unfortunate but this occurred the next night do to the snow fall. They are now asking for previous insurance policy numbers and phone records. I never thought the the company I hired(Geico) to protect me from things like this would potentially treat me as if I am the criminal. What are my rights and do I need an attorney? I mean should I wait to see what happens? Am I responsible for providing the burden of proof?( I only have evidence of a fast food drive through I was at shortly before). No one was hurt and I thought it was a minor fender bender so there is no police report.
Consult with a local insurance bad faith lawyer. This is a lawyer with special knowledge about the obligations insurers owe their insureds.
Many insurers have a internal unit - often referred to as a Special Investigations Unit (SIU) where certain types of claims are sent for investigation. I don't know if your claim has been sent to that unit.
A good lawyer will know how to deal with the SIU and help you with your claim. Good luck.
Thank you for your quick response! I will take your advice and consult with an Attorney.
I certainly understand how you feel. This insurance company like all others are out to make money. They don't make anything or really sell anything "tangible." They make money by taking in premium dollars (and investing that money) and not paying any out. They want to sell a policy and never have a claim on it. (Ultimately you want that also.) You turned out to be a nightmare for them. They took your money hoping to keep it, invest it and never pay you or anyone else on it. Instead right after they take your money, you make a claim!!!
They will have to pay you and depending on the circumstances of what they are doing and how long it takes for them to get this right, you may have a bad faith claim to bring. I recommend you consult with a personal injury lawyer that handles insurance bad faith claims. Some personal injury lawyers "dabble" in bad faith claims. I suggest you find someone who actively pursues these claims as a significant part of their practice.
If you feel this is the "best" answer or is "helpful," please indicate. Since I am limited to the information you provide, I cannot guarantee the accuracy of the answer. You should seek the advise of an attorney who can explore all aspects of your question. This communication does not form an attorney client relationship.
Look for an insurance bad faith attorney here on Avvo with the Find a Lawyer tool and do not talk with the insurance company further. Have all communications come from your attorney.
The information provided in this answer does not create an attorney-client relationship and is not considered to be legal advice.
Best bet is to get representation from a local insurance lawyer to battle it out with the company.
It may shock you to hear, but sometimes people drive without insurance and have an accident and then they go out and get insurance and claim that the accident actually happened a few days after it actually did so that the insurance will cover the damage. Given the unlucky timing of the accident, Geico wants to ensure that is not what happened in your case.
They want to know if you had similar coverage through another company before switching to Geico because that would show that their concerns are unfounded. If you had insurance coverage that would have covered this, there would be no reason for you to delay reporting the accident just so you could switch insurance and stick Geico with the bill.
You are responsible for cooperating with the insurance company if you want them to pay the claim. That is doubtless part of your policy language. If you do not cooperate, then they do not need to pay the claim.
Once the insurance company is satisfied that the information you reported is correct, they should pay the claim. They have a duty to act in good faith. If they act unreasonably in denying the claim, you should take to an attorney about pursuing a bad faith claim against the company.
You can reach Harkess & Salter LLC by phone or email. Stephen Harkess is an attorney licensed in the state and federal courts of Colorado. This answer is for general information only and does not create an attorney client relationship between Stephen Harkess or Harkess & Salter LLC and any person. You should schedule a consultation with an attorney to discuss the specifics of your legal issues.
Insurance companies generally have very broad rights in investigating a claim, and most likely there is a clause that calls for your full cooperation. If you fail to do so - such as not supplying requested information - GEICO may be able to deny your claim without ever judging it on the merits. You should read your policy very carefully. Your your state government should have some type of Department of Insurance that may assist you. Unless this is a claim for a lot of money, you will probable spend more on an attorney than resolving it with GEICO directly. A word to the wise - while GEICO, Progressive, etc. may be cheaper because they are online and there aren't any agents, this is the kind of stuff they pull. If you buy insurance though an "old school" company, you would have been able to take the claim to your friendly neighborhood agent who probably could have taken care of this for you. CAVEAT EMPTOR
I would call Rick Crane in Denver. He is an acknowledged expert and a font of knowledge in insurance coverage and bad faith matters as well as a former prosecutor. he will know what to do.
Sign up to receive a 3-part series of useful information and advice about personal injury law.
Post a free question on our public forum.Ask a Question
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Why Online Remote Computer Support?
- It saves you time
- It saves you money
- It's environmentally friendly
- No frustration of waiting for a technician
- No unnecessary call out fees
- No carbon footprint
How Can Remote Support Assist You?
Online, we can remove virus's, speed up your computer, connect multiple computers to each other, share printing, setup backups, emails, install programs for you, load missing drivers, etc, etc...
Yes, we can do all of that, plus a whole lot more. If you're experiencing any form of computer issue and have a working internet connection, remote support would potentially be the quickest and most cost effective method to resolve your problem. Up to 90 percent of all computer problems can be fixed remotely and if we cannot help you, there is no charge...
How Remote Support Works.
You simply download and install the remote support program at the bottom of this page by clicking on "CONNECT WITH A SPECIALIST". Once you start the session, you'll connect through to our offices where the session is accepted and remote support will commence. For security reasons, the remote support is completely one sided with only you being able to initiate a call.
No frills!..., No fuss!..., Computer support made simple!


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"Why did you choose this product?"
Lovely rug! Exceeded expectations....
Mohawk Home New Wave Picasso Wine (5' x 8')by Mohawk Home
ITEM#: 14118033This rug, embellished with Picasso-inspired shapes in warm tones, offers an upscale, contemporary look to any decor. Crafted of non-allergenic, stain- and fade-resistant nylon, it is soft underfoot and hassle free.
Let your stylish taste and love of abstract art show with this contemporary rug in rich warm tones, sure to add texture and depth to any room in your home. This alluring Picasso-inspired rug is machine tufted and made of resistant nylon for durability, as well as easy care and cleaning.
- Machine tufted
- Easy care and cleaning
- Action backing
- Primary materials: Nylon
- Pile Height: 0.406 inches
- Style: Contemporary
- Primary color: Gold
- Secondary colors: Burgundy, rust, dark green, tan
- Pattern: Abstract
Click here to view Mohawk Home Floor Care Essential Carpet Stain Remover
|Nylon, Synthetic
|Abstract, Border
|Contains Latex
|Weave Type
|Brown, Burgundy, Gold, Green, Rust, Tan
|Product Features
|Antimicrobial, Stain Resistant
|Rug Type
|5' x 8'
|1 year Limited Manufacturer
|Model Number
|10465 438 060096
|Country of Origin
|United States
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Customer Reviews
Most Recent
Great Buy
Lovely rug! Exceeded expectations.Read More
Sandra C.
I love this rug. The colors are deep and rich. It makes my small apartment look fantastic! It is not thick however the floor is carpeted.Read More
Floor Painting
This rug adds vibrant warm color to the room and certainly the floor. Good texture and very much represents the displayed photo on line. I would recommend it. Bear in mind not a thick pile which I did not want as it was for dining area.Read More
Ok. I bought for my living room but moved to laundry room. Just not good enough quality for a living roomRead More
The picture does not do it justice, Very happy with itRead More
Most Helpful
Picasso Rug
I wanted to purchase a colorful rug with various bright colors to go with my furniture already in the room. Also, I an an animal advocate with pets inside my house and I needed a little darker rug in case of any accidents in the house. This rug not only looks gorgeous with my furniture it doesn't show stains like my previous ones have. The quality of the materials looks rich and expensive. I was very very pleased with this purchase.Read More
Warm-looking and pretty
This carpet was about what I expected. It is thin, but I expected that considering the price. Still, I really love it--the colors and design are very pretty. It goes beautifully against my Brazillian hardwood floors in the family room and sets off my tan sofa, adding that touch of warm red that I think makes a great accent color. Since it's under the coffee table as a small area rug, the thin-ness is a benefit since you don't trip over a thick edge. Because of the rubberized backing, you don't need a non-slip pad underneath. Will hide all stains and pet issues, though I'll have neither. If you want thick and plush, this is not for you. I'm very happy with it!Read More
LOVE these rugs!!!
After reading some negative reviews about thses rugs, I was a little reluctant to order them, but when they went on sale for an additional *** off I decided to give them a try. Ordered two in the 5X8 size for the living room and the 2X8 runner for the adjacent dining room. Boy, am I glad I did! I LOVE the colors!! As several reviewers stated, the colors are brighter than the photos looked online but for me, that's a good thing! The rugs look beautiful against my dark hardwood floors. I didn't order rug pads for under them and they're not necessary b/c of the rubber backing. Since they arrived rolled up and bound with tight plastic wrap around the middle, it will take a few days for them to relax and lay flat but they are EXACTLY what I was looking for! I have 2 dogs so I didn't want to pay a fortune for area rugs; at the same time, while they're not super plush, they're definitely not cheap looking. I expect to get many compliments about them!Read More
cheap looking
this is a nylon rug. it says so in the description. I was hoping it would be as vibrant and classic looking as a wool or cotton hooked rug. it doesn't. to me it looks cheap. design is interesting but colors are somewhat drab. you can tell its an inexpensive nylon rug that was machine made. which is why I'm returning it. and I'll replace it with a wool rug from overstock that isn't that much more expensive than this one.Read More
Very nice
I found this rug to meet my need perfectly however the description about it being soft enough to lay on is a pipe dream of the writer. This is a very thin, very tough carpet. Consequently, the ends tend to curl up. The backing is rough so I am using a carpet pad to help all the way around and it works perfect for this carpet. I use this the rug for a sitting area with 2 chairs and a table. It does not get a lot of walking traffic. I think the colors are quite nice. They are muted in tone and the picture is really very good and matched what I received perfectly. I would recommend this carpet but not in high traffic areas and definitely with a carpet pad.Read More
Questions & Answers
nice colors
Size, looks & price
The reviews were very helpful and I like the colors on this rug
Matches living and dining rooms decor
It's great colors and I needed to bring a pop of color into my den area
I like the design and the color combination.
"Can this rug be taken outside and washed lightly to remove pet odor?"
Hello mj1010, This rug is machine washable. Have a great day!
"Does this pattern come in any other sizes"
Yes just search "Picasso Multi Contemporary Rug" on overstock.com and more sizes will come up.
"Am worried that this is thin and cheap. It's only .4" thick. My main question is what is an "action backing"? Does that mean it has a rubber back like a kitchen/bath rug? If so, that's just not right for my family room."
Action backing is secondary backing. In this item the action backing is woven polypropylene.
Shopping Tips & Inspiration
How to Protect Wood Floors with Area Rugs
The easiest way to protect wood floors from scratches and other damage is to decorate them with an area rug. Area rugs provide a protective barrier against furniture legs, foot traffic, and pets while adding style to any floor in the house.
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Get inspired by the hottest trends in living room furniture and decor. Shop our huge selection of living room furniture and decor at Overstock.com.
The Complete Rug Buying Guide
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Boho Chic Furniture and Decor Ideas
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Industrial Furniture and Decor Ideas
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Shipping & Returns
Contact Information
Usually leaves our warehouse in 1-2 business days. *
Shipping Information:
Our standard shipping method is ground shipping. We also offer expedited shipping for most items. Your final
shipping cost and estimated delivery timeframe will be available at checkout.
Standard shipping details
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Items must be returned in new or unused condition and contain all original materials included with the shipment. More Details
FINAL SALE EXCLUSION: Items marked as FINAL SALE are not returnable unless the problem you experience is the result of our error.
For your protection, all orders are screened for security purposes. If your order is selected for review, our Loss Prevention Team may contact you by phone or email. There may be a two business day delay to process your order.
** Most Oversize orders are delivered within 1-4 weeks. Some orders may take 6 weeks to be delivered.


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Greek6th - 5th Century B.C.Bronze
RomanCirca 1st Century B.C. - 1st Century A.D.Marble
RomanCirca 1st Century B.C. - 1st Century A.D. Glass
Roman1st - 2nd Century A.D. Stone
GreekCirca 6th Century B.C.Pottery
Attic Black-Figure Olpe by the Painter of Vatican G49.
Funerary masks were extremely important in ancient Egyptian culture.
The finely detailed lion has a recumbent head, turned sharply to the left and facing forward...
The egg shaped body with a small low foot, the handle in the form of a monkey...
An amphora (plural amphorae) is a vase-shaped container with two handles and a neck...
The Greek myth of Ariadne offers hope to all who have felt the sting of loves rejection.s rejection.
Carved in high relief with two brothers joining hands, one holding a cluster of grapes and wearing a himation with a beaded hem along the collar...
This item was most likely utilized in covering the entire floor of a small room.
This oil lamp is finely molded, with a good symmetry of both shape and artwork.
Large Roman mosaic with a geometric design of cicles and diamonds.
We are ancient art sellers who are always looking to buy art that has been legally acquired. If you have any authentic antiquites, artifacts, or ancient art for sale.
Aphrodite Ancient Art is an established seller of ancient art and antiquites, with a gallery in New York City on 5th Avenue 'Museum Mile', dealing in fine museum quality antiquities.
For over a decade, Aphrodite Ancient Art has offered some extraordinary pieces to antiquities and artifacts collectors, dealers, and museums worldwide. Everything we offer is categorically guaranteed authentic. All of our art has been legally acquired from generations of collectors spanning the globe.
Presently for sale are Greek, Egyptian and Roman, Near Eastern, Islamic, and Byzantine works of tremendous cultural significance. From pottery to bronze, our compendium of ancient antiquities is as diverse as the mediums it consists of. We are among the world's top ancient glass dealers and have amassed a large and comprehensive selection, exemplary of many different styles, production techniques, and uses.
Whether you are a beginning or advanced collector, we gladly and freely offer information and advice regarding investing in ancient art. Our collection caters to people at all price points, and the more typical pieces as well as the recherché can be more easily accessible as well as expensive. Follow your interests: view the works we have on display and don't hesitate to ask any questions. We believe you're destined to acquire pieces that really speak to you. Aphrodite Ancient Art will strive to bridge the gap between you and those artifacts.
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Amazing Magic of Wes Booth
955 Corinna Rd
Designing Minds Salon
85 Mountain Rd
FYP.INC Independent Business Associate
919 Dexter Rd. St. Albans, Me.
Nre Transparent
437 Nokomis Rd
Shared Living
468 Corinna Rd
St Albans Auto Repair
20 Main St
Sunrise Restaurant & Bakery
122 Hartland Rd
30 Main St


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| Next
Polyform S Series Buoy 1.6 Gallon White
Polyform US
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a The adaptable S Series S-1 buoy is an excellent choice for multipurpose applications. Use as a fender or mooring buoy for small boats and jet skis. Its compact size is perfect when storage space is limited. The S Series' elongated design allows it so sit higher in the water than an ordinary round buoy. Replace the "bleach bottle" when small pot and trap fishing with a durable, distinctly visible marker buoy. Water sport associations and competition enthusiasts combine the S-1 "boat guide" with the Polyform WS-1 Slalom Buoy as a complete course-marker package. For high-performance or leisure water sports, choose the versatile S Series S-1 for your small fender and buoy needs.
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6 x 15 in.
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text:
August 11
by: ASRM Office of Public Affairs
Orginally published in ASRM Bulletin Volume 17, Number 28
Comments attributable to James Toner, MD, President of SART and Rebecca Z. Sokol, MD, MPH, President of ASRM.
James Toner, MD, SART President remarked, "We appreciate Dr. Kushnir and his colleagues' sharing their analysis comparing the outcomes of cycles using frozen donor eggs with outcomes of fresh donor egg cycles. This year, for the first time, SART included frozen donor egg cycles in its National Report, enabling this large-scale, nationwide comparison to be done.
While frozen donor egg cycles resulted in a somewhat lower percentage of live births than fresh in this national report, this outcome is not unexpected since recipients of frozen eggs typically get fewer eggs for a treatment half as expensive as traditional donor egg with fresh eggs.
As the researchers pointed out, egg freezing has only recently been widely adopted, and these results show how effective this approach can be for those needing donor eggs. It is not yet known whether these reassuring outcomes will be seen in woman in their 30s and 40s.
A more complete understanding of these outcomes will take time. Eggs may remain frozen for many years and the eggs frozen from a single retrieval may be thawed and fertilized singly or in pairs, delaying knowledge of the full cycle outcome for years."
Rebecca Z. Sokol, MD, MPH, ASRM President added, "There are a host of reasons that a patient may need to freeze her own eggs. When faced with chemotherapy or other cytotoxic treatment, egg freezing is the best hope of preserving a woman's fertility. If sperm cannot be obtained at the time of a fresh IVF cycle, egg freezing can salvage the cycle.
Frozen egg banking gives patients access to a wider field of donors and provides greater flexibility for both donors' and recipients' scheduling and coordination, advantages that may outweigh slightly lower success rates for some patients.
We need to recognize that cryopreservation is a procedure that requires specific technical expertise and proficiency. While great improvements in technique and outcomes have been made in recent years and we expect to see more in the future, patients should be cautioned that putting their eggs into storage today is no guarantee of having a baby tomorrow."
Sean Tipton
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text:
Jayden Woods is the author's pen name. Jayden is a writing graduate from the University of Southern California and has also lived in Tennessee and Missouri. Jayden wrote nine novels and several screenplays before releasing any work to the public, and began by posting the online webserial, "Lost Tales of Mercia." The historical fiction novel, "Eadric the Grasper," launches October 5, 2010.
Lost Tales of Merciaby Jayden Woods
A mother defies an ealdorman for the sake of her son. A boy becomes king at eleven years of age through the scandalous death of his brother. A young girl takes desperate measures to manipulate the king's court. An eccentric noble gives a young swineherd a priceless gift in the form of education. An orphaned woman joins the Viking prince to pursue her revenge.
These are only the first five stories of the ten Lost Tales of Mercia. They introduce the characters who will fight, love, and betray each other until the rightful king takes the throne of Engla-lond. These stories serve as a complement to the novel, "Eadric the Grasper." Though available online as individual ebooks, this is the full compilation of the ten tales in printed format.
Product Details
- 9781453799925
- Publisher:
- CreateSpace Publishing
- Publication date:
- 10/21/2010
- Pages:
- 232
- Product dimensions:
- 5.50(w) x 8.50(h) x 0.49(d)
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This is a collection of short stories, which you can download separately for free as well. Which I did and it looks great on my NookColor haha Each story is about a different person all taking place during the years of an ongoing war. Royalty, Vikings, peasants, children- These stories cover all kinds of people and they are ALL interesting to read about, especially because they eventually tie in together. I want MORE!
If you enjoy historical fiction then this the series for you. These tales are wonderfully written and the characters are so vivid i feel as if i am watching a movie.
The tales seem unconnected at first, and at the end of each I felt frustration at being left hanging. I began each new tale confused about where these new characters fit into the last tale, and read on, hoping for more about the last tale. By the end of the book, I saw a few connections between the tales and desperately wanted more! I was satisfied with more in Eadric the Grasper and Godric the Kingslayer - both wonderful full stories stemming from the tales and beginning the work of connecting the tales. I highly recommend the these tales, as well as the Sons of Mercia series that ties them together!
These interwoven tales are quite enjoyable. I couldn't put it down. Although fictionalized, these true to history events come alive in all their glory. The political implications of dry historical facts brings Mercia to life. So good that I had to buy another in the series.
I highly recommend this book (and the follow-up Eric the Grasper) as "required reading" for dark ages reading buffs!
i loved this it wass awsome
The author wove the ten stories into a great read. It all made sense and gave you a great feel of the period. He stories reminded me of the novels that Bernard Cornwell wrote covering this period of English history. I recommend this book.
The stories should have been complete. All you get are cliffhangers.
Love the short stories. I find that when I finish one I quickly go to the next. The stories are short enough to read easily but keep the interest up. Can't wait to Eadric the Grasper.


text:
A different immunologic profile characterizes patients with HER-2-overexpressing and HER-2-negative locally advanced breast cancer: implications for immune-based therapies
DOI: 10.1186/bcr3060
© Muraro et al.; licensee BioMed Central Ltd. 2011
Received: 9 August 2011
Accepted: 23 November 2011
Published: 23 November 2011
The clinical efficacy of trastuzumab and taxanes is at least partly related to their ability to mediate or promote antitumor immune responses. On these grounds, a careful analysis of basal immune profile may be capital to dissect the heterogeneity of clinical responses to these drugs in patients with locally advanced breast cancer undergoing neoadjuvant chemotherapy.
Blood samples were collected from 61 locally advanced breast cancers (36 HER2- and 25 HER2+) at diagnosis and from 23 healthy women. Immunophenotypic profiling of circulating and intratumor immune cells, including regulatory T (Treg) cells, was assessed by flow cytometry and immunohistochemistry, respectively. Serum levels of 10 different cytokines were assessed by multiplex immunoassays. CD8+ T cell responses to multiple tumor-associated antigens (TAA) were evaluated by IFN-γ-enzyme-linked immunosorbent spot (ELISPOT). The Student's t test for two tailed distributions and the Wilcoxon two-sample test were used for the statistical analysis of the data.
The proportion of circulating immune effectors was similar in HER2+ patients and healthy donors, whereas higher percentages of natural killer and Treg cells and a lower CD4+/CD8+ T cell ratio (with a prevalence of naïve and central memory CD8+ T cells) were observed in HER2- cases. Higher numbers of circulating CD8+ T cells specific for several HLA-A*0201-restricted TAA-derived peptides were observed in HER2+ cases, together with a higher prevalence of intratumor CD8+ T cells. Serum cytokine profile of HER2+ patients was similar to that of controls, whereas HER2- cases showed significantly lower cytokine amounts compared to healthy women (IL-2, IL-8, IL-6) and HER2+ cases (IL-2, IL-1β, IL-8, IL-6, IL-10).
Compared to HER2- cases, patients with HER2-overexpressing locally advanced breast cancer show a more limited tumor-related immune suppression. This may account for the clinical benefit achieved in this subset of patients with the use of drugs acting through, but also promoting, immune-mediated effects.
KeywordsBreast cancer neoadjuvant therapy HER2 trastuzumab antitumor immune responses tumor-associated antigens
Preoperative or neoadjuvant chemotherapy (NC) is currently considered to be the standard of care for locally advanced and inoperable breast cancer. One of the main advantages of this approach is the reduction of tumor size, which increases the possibility of performing smaller resections of operable tumors with better cosmetic outcomes [1, 2]. Other potential benefits of NC include an early assessment of response to chemotherapy and the possibility of obtaining prognostic/predictive information, based on the pathologic response to therapy [3].
Although breast cancers that overexpress human epidermal growth factor receptor-2 (HER2) are characterized by a poor prognosis [4, 5], higher rates of complete responses are currently achieved in HER2+ patients by standard chemotherapy, mainly in association with trastuzumab [6, 7], in comparison with HER2- patients. Like other monoclonal antibodies used in anticancer therapy, the activity of trastuzumab is largely dependent on immuno-mediated mechanisms. In fact, besides triggering antibody-dependent cytotoxicity (ADCC), trastuzumab also enhances HLA class I-restricted presentation of endogenous HER2 antigen via the proteasome pathway, and sensitizes HER2-overexpressing tumors to killing by MHC class I-restricted HER2-specific cytotoxic T lymphocytes (CTLs) [7]. Intriguingly, other drugs used in NC regimens have also been shown to enhance antigen-specific immune responses in both in vitro and animal models. In particular, taxanes have immunostimulatory effects against tumor cells and suppress cancer not only through inhibition of cell division [8, 9]. Indeed, hosts immune functions are highly enhanced after docetaxel treatment [10], and paclitaxel plays a positive role in controlling tumor growth, probably through the induction of IL-8 [8]. Furthermore, taxanes induce macrophage-mediated tumor death, stimulate the production of pro-inflammatory cytokines (TNF-α, IL-12, and IL-1), and increase lymphokine activated killer (LAK) cell and natural killer (NK) cell antitumor activity [10, 11].
Given the evidence that tumor cells may be immunogenic, more than 60 TAAs have been identified and, as observed for other tumors, breast cancer cells were also shown to express TAAs [12, 13]. Moreover, convincing data demonstrate that spontaneous antitumor responses to TAAs may harness host's immune system to fight against cancer, underscoring the need of a retained or only minimally compromised immunological proficiency particularly in patients treated with chemotherapeutic regimens including immunomodulating drugs. Nevertheless, only limited information is available on the extent of spontaneous T cell responses to breast cancer-associated antigens in patients with locally advanced tumors.
Considering that breast cancer patients may show different types and extent of tumor-related immune dysfunctions [11, 14, 15], we reasoned that the efficiency of the host immune system could influence the responses to current NC regimens. Therefore, in the present study, we have carried out an extensive immunologic profiling of patients with locally advanced breast cancer at the time of diagnosis, as a first step towards a better understanding of the possible role of antitumor immune responses in mediating the clinical outcome of NC. The results presented herein demonstrate that patients with HER2+ and HER2- breast cancer have a different basal immunologic profile. In particular, our data are consistent with a more limited tumor-related immune suppression in patients with HER2-overexpressing tumors, an observation that may at least in part account for the clinical benefit achieved in this subset of patients by drugs acting through immune-mediated effects.
Materials and methods
Patients and healthy donors
Patients' characteristics
Mean age, years
Hormone receptor status 1
ER+ and PgR+
ER+ and PgR-
ER- and PgR-
ER- and PgR+
CISH/FISH not amplified
CISH/FISH amplified
Tumor size
18 (2 multifocal)
26 (2 multifocal)
7 (1 multifocal)
10 (1 multifocal)
Clinical lymph node involvement
Grading 2
Peptide selection and synthesis
A total of 13 immunogenic HLA-A*0201 nonamer (9-mer) peptides, derived from different breast cancer-associated antigens (survivin, mammaglobin-A, HER2, mucin-1, taxol-resistence associated gene 3, and bcl-XL) were selected for the study. The HLA-A*0201-restricted Flu matrix 1 (M158-66) peptide (GILGFVFTL) was used as the positive control. All peptides were produced by fluorenylmethoxycarbonil synthesis (Primm, Milan, Italy) and purity (>95%) was determined by reverse-phase high-performance liquid chromatography and verified by mass spectral MALDI-TOF analysis. Peptides were dissolved in DMSO at a concentration of 2.5 mg/ml and stored at -70°C until use. Work stocks for each peptide were prepared in PBS at a final concentration of 500 μg/ml and stored frozen.
Flow cytometry
The following fluorescent-conjugated monoclonal antibodies were used: α-CD3 fluorescein isothiocyanate (FITC) or phycoerythrin-texasred (ECD; mouse immunoglobulin (Ig) G1, clone UCHT1), α-CD4 phycoerythrin-cyanine5 (PC5; mouse IgG1, 13B8.2), α-CD8 phycoerythrin-cyanine7 (PC7; mouse IgG1, SFCI2IThy2D3), α-CD16 FITC (mouse IgG1, 3G8), α-CD19 FITC (mouse IgG1 k, J3-119), α-CD25 ECD (mouse IgG2a, B1.49.9), and α-CD45RA ECD (mouse IgG1, 2H4LDH11LDB9) all from Beckman Coulter (Fullerton, CA, USA); α-CD56 phycoerythrin (PE; mouse IgG1 k, B159) and α-CD197 PE (CCR7, rat IgG2a k, 3D12) purchased from BD Biosciences (Becton Dickinson, Franklin Lakes, NJ, USA); α-CD4 PC7 (mouse IgG1 k, RPA-T4), α-CD127 PC5 (mouse IgG1, eBioRDR5), and α-FoxP3 PE (Rat IgG2a k, PCH101) from eBioscience (San Diego, CA, USA). Properly labelled isotypic antibodies were used as negative controls. All antibodies were used in an appropriate volume of 10% rabbit serum (Dako, Glosdrup, Denmark) and PBS (Biomerieux, Marcy l'Etoile, France) to reduce nonspecific signal. Intracellular FoxP3 was determined using the eBioscience FoxP3 Staining Buffer Set (eBioscience, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, after surface molecules staining, cells were fixed and permeabilized with fixation/permeabilization buffer for 30 minutes at 4°C, washed twice, and labelled with FoxP3 antibody in the presence of 2% rabbit serum in PBS at 4°C for at least 30 minutes and, after two washes, cells were re-suspended in PBS with 1% paraformaldehyde. At least 5 × 104 cells for surface markers and 1 × 106 cells for intracellular staining were acquired. Cytofluorimetric analysis was performed with a Cytomics FC500 (Beckman Coulter, Fullerton, CA, USA) and data were analyzed with CXP software (Beckman Coulter, Fullerton, CA, USA).
IFN-γ ELISPOT assay
The interferon (IFN)-γ release enzyme-linked immunosorbent spot (ELISPOT) assay was performed using a commercial kit (Human IFN gamma ELISPOT; Thermo scientific, Rockford, IL, USA), according to manufacturer's instructions. The assay was carried out using autologous peptide-pulsed monocytes as antigen presenting cells (APCs) and isolated CD8+ T lymphocytes as responders. Monocytes, isolated by a two hour plastic adherence step from patient's PBMCs, were loaded with 10 μg/ml of each 9-mer peptide in complete medium, supplemented with 5 μg/ml of human β2-microglobulin, and incubated for two hours at 37°C with 5% CO2. Purified effectors were obtained by immunomagnetic enrichment protocols using the human CD8+ T cell isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany), and then cultured with peptide-loaded monocytes (50,000 cells/well) at 1:1 effector:target ratio. FLU M158-66 and unstimulated monocytes were used as positive and negative controls, respectively. Cells were seeded onto ELISPOT capture plates in triplicates and incubated for 48 hours at 37°C with 5% CO2. All plates were evaluated by a computer-assisted ELISPOT reader (Eli.Expert, A.EL.VIS GmbH, Hannover, Germany). The number of spots in negative control wells (range of 0 to 5 spots) was subtracted from the number of spots in stimulated wells. Responses were considered significant if a minimum of five IFN-γ producing cells were detected in the wells.
Cytokine detection
Levels of IL-1α, IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, TNF-α, and granulocyte macrophage colony-stimulating factor (GM-CSF) were evaluated using the SearchLight® multiplex arrays (Food and Drug Administration approved, Aushon Biosystems, TEMA Ricerca, Bologna, Italy) according to the manufacturer's instructions. Briefly, custom human 8-plexarray and human 1-plexarray (for GM-CSF detection) with pre-spotted cytokine-specific antibodies were used. Standards or pre-diluted samples were added in duplicate and, after one hour of incubation at room temperature and three washes, biotinylated antibody reagent was added to each well. After 30 minutes incubation at room temperature and three washes, block solution was added to stabilize the signal. The addition of Streptavidin-HRP Reagent and SuperSignal® Substrate, and the acquisition of luminescent signal with a cooled CCD (Charge Coupled Device) camera, together with data analysis and processing, were performed by TEMA Ricerca laboratories' customer service (Bologna, Italy).
Transforming growth factor (TGF)-β1 serum levels were assessed through DRG TGF-β1 ELISA (DRG Instruments GmbH, Marburg, Germany) under instructions. Pre-diluted samples and standards underwent appropriate acidification and neutralization before testing. Briefly, pre-treated standards, controls and samples were dispensed into wells in duplicate and plates were incubated overnight at 4°C. After three washes, antiserum was added to wells and incubated for 120 minutes at room temperature, plate was rinsed three times and anti-mouse biotin (enzyme conjugate) was dispensed and incubated for 45 minutes. After three washes, enzyme complex was added to wells, then plates were incubated 45 minutes and washed three times. After the addition of substrate solution for 15 minutes, the reaction was stopped and the adsorbance at 450 ± 10 nm was determined with a microtiter plate reader (Bio-Tek Instruments, Winooski, VT, USA). As there is an extremely variable range of normal values reported in the literature, healthy women serum levels were taken as references.
Considering that the diagnostic biopsy is not fully representative of the whole tumor mass, the lymphoid infiltrate was investigated in 40 primary advanced breast carcinomas from a separate series of patients who underwent surgical resection during the past decade at our institution. Twenty cases were HER-2+ (3+) and 20 HER-2- (0 or 1+). All specimens were routinely fixed in 10% buffered formalin, embedded in paraffin and then stained with H&E for histological examination. For immunohistochemical analyses, 2 to 3 µm serial sections of primary tumors were processed with automated immunostainer Benchmark XT (Ventana, Tucson, AZ, USA), and staining was carried out with the following antibodies: CD8 (clone SP57, Ventana Medical System, Tucson, AZ, USA); FoxP3 (clone 259D/C7, BD Pharmingen, Franklin Lakes, NJ, USA) diluted 1:100 e TiA-1 (clone TiA-1, Bioreagents, Golden, CO, USA) diluted 1:100. Nuclear counterstaining was accomplished with Harris' hematoxylin. Omission of the primary antibody was used as a negative control. The results for staining were evaluated with reference to the number of unequivocally stained lymphoid cells. Ten randomly chosen representative microscopic fields were counted at 40x original magnification.
Statistical analysis
Chi-square test was used to compare hormone receptor (HR) expression and grading within HER2- and HER2+ populations. Data obtained from multiple independent experiments were expressed as mean and standard deviation for immunophenotypic analysis and ELISPOT assays; cytokine box plots were obtained with SigmaPlot. The Student's t test for two tailed distributions and the Wilcoxon two-sample test were used for the statistical analysis of the data. Odds ratio and 95% confidence intervals in multivariate analysis were performed to assess the possible influence of clinico-pathological variables on the immunological correlations observed: immunological variables (cytokine levels) were divided into quartiles according to their concentrations and then stratified for HR expression (estrogen receptor (ER)- progesterone receptor (PgR)- and ER+ and/or PgR+), and tumor grading (G2 or G3). Wilcoxon rank-test was used to compare the distribution of intratumor CD8+ and FoxP3+ cells between HER2+ and HER2- cases. Results were considered to be statistically significant when P ≤ 0.05 (two-sided).
HER2+ and HER2- patients exhibit a different distribution of circulating and intratumor immune cells
When the CD3+ population was considered separately, HER2- patients showed significantly higher percentages of CD8+ T cells (P = 0.028; data not shown) and a lower CD4+/CD8+ ratio (Figure 1d; P = 0.046) when compared with HER2+ cases. Because of the different contribution of memory subsets in mediating antitumor immune responses [17–19], the differentiation state of T cell was investigated through the combined analysis of the chemokine receptor CCR7 and the CD45RA isoform, to distinguish CCR7+CD45RA+ naïve, CCR7+CD45RA- central memory (CM), CCR7-CD45RA- effector memory (EM), and CCR7-CD45RA+ terminally differentiated (Temra) cells [20]. Although the three studied groups showed a similar distribution of memory CD3+ T cell subsets (not shown), separate analysis of the CD4+ and CD8+ compartments disclosed remarkable differences. In fact, compared with controls, HER2+ patients carried a higher percentage of CM CD4+ T cells (P = 0.003), whereas HER2- cases showed significantly higher numbers of CM CD8+ T lymphocytes (P = 0.022). Moreover, a higher percentage of EM CD4+ cells (P = 0.023), at the expense of the CM subset (P = 0.023), was found in HER2- cases compared with HER2+ patients (Figure 1f). Finally, the two groups of breast cancer patients showed a completely different memory sharing among CD8+ cells, with a prevalence of naïve (P = 0.002) and CM cells (P = 0.005) in HER2- cases and higher percentages of EM (P = 0.005) and Temra cells (P = 0.012) in HER2+ patients.
Several studies argued the unfavorable involvement of circulating regulatory T cells (Tregs) in cancer progression, demonstrating the presence of increased numbers of CD4+CD25highFoxP3+ especially in metastatic cancers [21]. Considering that this immunophenotypic characterization is unsuitable for uniquely defining this specialized T cell subset, we used IL-7 receptor (CD127) down-regulation as a further feature indicative of suppressive functions [22] and identified Tregs as CD4+CD25highCD127lowFoxP3+ cells. The analysis showed that total numbers of so determined Tregs were not significantly different between patients and controls. Nevertheless, considering the two groups of patients separately, HER2- exhibited a significantly higher percentage of circulating Treg cells (P = 0.02) when compared with healthy donors (Figure 1e).
HER2+ patients display enhanced CD8+ T cell responses to different TAA-derived epitopes compared with both HER2- patients and healthy donors
Library of immunogenic peptide epitopes used to evaluate CD8+ T cell responses to breast-cancer-associated antigens
HER2+ patients show similar serum cytokine profile in respect to donors
Odds ratio (OR) and 95% confidence interval (CI) adjusted for hormone receptor expression and tumor grading according to cytokines levels in HER+ and HER- patients
Cytokines level quartiles1
N. pos/neg2
6.3 (0.7-53.2)
33.6 (2.3-494.6)
55.4 (4.1-746.0)
N. pos/neg2
15.7 (1.6-150.5)
10.3 (1.2-92.4)
31.8 (3.5-289.6)
N. pos/neg2
3.8 (0.5-31.0)
8.2 (1.0-65.4)
21.9 (2.6-185.8)
N. pos/neg2
3.7 (0.5-26.0)
5.6 (0.7-42.3)
11.7 (1.4-95.5)
N. pos/neg2
0.3 (0.04-2.6)
1.6 (0.2-12.1)
9.6 (1.5-59.0)
Evidence accumulated so far indicates that the immune system can influence the initiation and development of cancer and it is widely believed that T lymphocytes represent the most potent antitumor effector cells. In this respect, there is an urgent need to develop therapeutic approaches able to preserve or only minimally impair immune functions since ADCC-promoting therapeutic antibodies, such as trastuzumab, and cancer vaccines are being increasingly used as adjuvant and neoadjuvant treatment modalities [23]. Furthermore, also the therapeutic efficacy of some "conventional" drugs, such as doxorubicin and paclitaxel, involves immunomediated mechanisms [11, 24]. On these grounds, we considered it clinically relevant to extensively investigate at diagnosis the immunological profile of locally advanced breast cancer patients who are candidates for NC including immunomodulating drugs. The present study provides baseline immunological data that may constitute a reference for an informative monitoring of immune responses during NC (ongoing study). Provided the feasibility of monitoring antitumor responses during therapy and considering that sera from breast cancer patients should represent a valuable discovery tool to identify potential targets involved in breast cancer progression [25], we focused mainly on peripheral blood as the easiest accessible way to detect and measure immune-changes.
It is worth considering that most studies reporting analyses of systemic immunologic parameters in breast cancer patients included extremely variable series of cases, thus obtaining wide ranges of values and often conflicting results. Our study does not suffer from this limitation, being based on a relatively homogeneous group of patients including only locally advanced cancers and excluding metastatic patients, which are the main contributors to outliers [26, 27].
Previous data reported a significant increase in circulating B lymphocytes and NK cells in breast cancer patients in comparison with control groups [14], with a lower total number of T lymphocytes [15]. Interestingly, in our study, HER2+ patients retained a normal distribution in NK cells, and T and B lymphocytes if compared with healthy donors, whereas HER2- cases displayed lower percentages of T cells and higher numbers of NK and, to a lesser extent, of B cells. The increased NK cell numbers and activity reported in pre-treated patients were related to time to treatment failure [14] and were proposed to be the result of activation of the innate immunity by the tumor or dependent on a defective regulation of NK cells in these patients [28]. Conversely, Dewan and colleagues observed significantly lower NK cell activity in PBMC from breast cancer patients, as compared with that of healthy individuals. Intriguingly, this defect was more pronounced in HER2- breast cancer patients [15], suggesting an underlying NK dysfunction in this subgroup. Further, we noticed that HER2+ patients showed an increased CD4/CD8 ratio with respect to HER2- cases, a feature previously associated with a better chance of responding to NC [14]. However, opinions regarding which T-cell subset provides the best tumor protection, especially among memory sub-populations, are still controversial. Indeed experimental evidence suggests that central (TCM) and effector (TEM) memory T cells can each confer a protective advantage [17–19], with TCM providing a reservoir of antigen-specific T cells, ready to expand and replenish the periphery upon secondary challenge, and TEM displaying a more activated phenotype capable of granzyme B and perforin expression, IFN-γ secretion, and tumor-specific killing in vitro [17]. Compared with healthy donors, our data disclosed a higher percentage of CD4+ TCM (CCR7+CD45RA-) in HER2+ patients and higher numbers of CD8+ TCM in HER2- cases. This observation may suggest the existence, in both groups, of an active, though predominantly memory T-cell driven, antitumor response which may benefit and respond to recall antigens from a cancer vaccine. Interestingly, in our study the main variations in memory subsets distribution were found between HER2- and HER2+ patients. Although the CD4+ T cell population of HER2+ cases disclosed a favorable shift to the TCM phenotype, in these same patients CD8+ T lymphocytes revealed a predominance of TEM and terminally differentiated cells. In contrast, HER2- patients exhibit mainly CCR7+ CD8+ T cells (naïve and TCM). It should be considered that peripheral CD8+ T cells expressing effector functions against viral [29, 30] or tumor antigens [31] are almost uniformly CCR7- and are endowed with full effector capacities, far greater than naïve or TCM cells. Moreover, as the differentiation to terminal effector cells is related to increased cytolytic potential of CD8+ T cells, we hypothesize that the extent of maturation might be due to an effective antitumor response [18].
Pre-existing T cell responses to TAA have been reported in patients with solid tumors [32]; however, these responses usually involve a low frequency of antigen-specific T cells, not detectable in the majority of patients [18]. In this regard, literature data on circulating tumor antigen-specific T cells in breast cancer patients are still conflicting, probably because of the predominant focus on single epitopes [18]. Circulating T cells able to recognize CD8+ epitopes of HER2 [12], MUC-1 [33], mammaglobin-A [34], Trag-3 [35], survivin [36], or bcl-xL [13] have been described in distinct papers, but the evaluation of multiepitopic antitumor responses is still lacking. We therefore assessed the amount of IFN-γ-secreting CD8+ T cells specific for a broad spectrum of HLA-A*0201 peptides derived from Her2, muc-1, mam-A, trag-3, survivin, and bcl-xL. Notably, we found increased IFN-γ release to all screened epitopes in the global cohort of patients if compared with healthy donors, demonstrating the existence of spontaneous T cell responses against multiple TAA in locally advanced breast cancer patients. The ability to stimulate the generation of antitumor CD8+ T cells seemed to be more pronounced in HER2+ cancers, especially towards Her2-, trag-3-, muc-1-, and bcl-xL-derived epitopes. This peculiarity may be useful in the design and optimization of vaccine strategies, which could take advantage of host's pre-existing antitumor immune response. Moreover, the increased numbers of TAA-specific circulating CD8+ T cells characterizing HER2+ patients may positively contribute to the clinical efficacy of trastuzumab, which is able to sensitize HER2-overexpressing tumors to the killing by HER2-specific CTLs [7], and may enhance the antigen-specific immune responses promoted by doxorubicin and paclitaxel [37]. Our findings at the systemic level are also consistent with the demonstration of a significantly higher prevalence of CD8+ T lymphocytes infiltrating HER2+ tumors that could contribute to a better clinical outcome [38]. This suggests that HER2 overexpression may be associated with enhanced immunogenicity of tumor cells and/or with a less immunosuppressive microenvironment. Further characterization of the activation state of lymphocytes infiltrating these tumors is, however, required to draw definitive conclusions in this respect.
It is well recognized that tumors may down-regulate the immune response to tumor antigens by inducing several immune suppressor mechanisms, including Treg recruitment. Increased numbers of Tregs have been correlated with greater disease burden and poorer overall survival [39]. In particular, Treg cells are augmented in the peripheral blood and within the tumor microenvironment in patients with breast carcinomas [40]. Our analysis of FoxP3 expression in intratumor lymphocytes disclosed significantly higher prevalence of FoxP3+ cells in HER2+ tumors. This may be a homeostatic consequence of the higher content of infiltrating CD8+ T cells detected in these cancers, or may reflect an active local recruitment of Tregs. It is worth considering in this respect that conflicting results have been reported with regard to Treg frequencies in HER2- versus HER2+ breast cancers [27, 41], discrepancies that could be due in part to tumor staging, but also to the different and often partial phenotypic markers used to identify regulatory T cells. This suppressor cell subset is often identified as CD4+CD25high cells [27] or as CD4+FoxP3+ lymphocytes [41], even if these markers are unable to uniquely define a regulatory T cell phenotype. Therefore, the use of FoxP3 as a single immunohistochemical marker to identify Tregs may have overestimated the number of Tregs in the HER2+ subgroup. In fact, this approach can not discriminate true FoxP3+ Treg cells from T lymphocytes activated by local stimuli and therefore transiently expressing FoxP3 without being endowed of immunosuppressive functions. To overcome these limitations, we have bona fide considered as circulating Tregs only cells expressing the CD4+CD25highCD127lowFoxP3+ phenotype, as the down-regulation of the IL-7 receptor (CD127) is associated with suppressive functions [22]. This extended phenotypic definition did not disclose significant differences in Treg distribution between the whole group of breast cancer patients and controls, but it revealed higher numbers of circulating Tregs in HER2- patients with respect to donors. Tregs exert their suppressor activity by inhibiting T cell proliferation, NK cell-mediated cytotoxicity [42], and TAA-specific immunity [43]. On these grounds, the nearly physiological number of circulating Tregs displayed by stage II and III HER2+ breast cancer patients may imply a favorable background for NK-involving therapy such as monoclonal antibodies (trastuzumab), and may further benefit from the spontaneous enhanced antitumor T cell responses.
The likely retained immune proficiency of HER2+ patients is supported by an apparently unchanged cytokine profile, as sharpened by the comparison with serum cytokine levels of healthy women, which displays no significant differences. It is widely accepted that solid tumors are associated with a pathologic shift toward the T-helper type 2 cytokine pattern; whereas T-helper 1-induced inflammation inhibits tumor growth. In breast cancer patients, depressed serum levels of IL-2, GM-CSF, IFN-γ and enhanced TNF-α and IL-6 amounts were reported in comparison with controls [11] and some of these immune dysfunctions are also present in early-stage tumors. In our study, we noticed significantly lower levels of IL-2 and IL-8, but also of IL-6 in HER2- patients with respect to healthy women. Increased serum levels of IL-6 were previously reported in progressive recurrent breast cancer patients [44], especially in the presence of metastasis [26], conversely no metastatic cases were enrolled in our cohort. Interestingly, the comparison of cytokine layouts between HER2- and HER2+ patients disclosed pathogenically relevant differences between the two groups. In particular, HER2- patients showed lower levels of IL-2, previously associated with relapse of the disease [45, 46], reduced amounts of the pro-inflammatory cytokines IL-1β and IL-6 (repressors of in vitro cell cycle progression) [47], and reduced levels of IL-8, recently reported also in the in vitro comparison of HER2- and HER2+ breast cancers cell lines and in serum samples from metastatic breast cancer patients [25]. Finally, the pleiotropic cytokine IL-10, which may exert tumor-promoting activity or considerable antitumor effects, at low and high concentrations, respectively [46], was detected at lower amounts in HER2- patients. On the other hand, the higher levels of IL-2 in HER2+ patients may be consistent with an activation of T cells by TAA-derived peptides [48]. Notably, the differences in the cytokine levels observed between HER2- and HER2+ cases were independent of the clinical-pathological features showed by the two cohorts of patients (Table 3).
The different immunologic profile of patients with HER2- and HER2+ tumors highlights the importance of considering them as two distinct populations not only with regard to tumor characteristics, but also concerning their immune status. Our analysis, however, may show some limitations mainly due to the quite large number of immunological factors considered. This multiparametric approach has considerably restricted the global case series, thus limiting the possibility to make comparisons of adequate statistical power between subpopulations of HER2- and HER2+ patients. Larger series should be therefore investigated to conclusively rule out the possible influence of distinct clinico-pathological variables on the immunological correlations observed. Moreover, the research of TAA memory T-cell responses was confined at the peripheral immune compartment; an in situ comparative survey is needed to confirm our data. An interesting issue that needs further investigation is the assessment of whether the higher percentage of effector memory CD8+ T cells observed in HER2+ patients correlates with the enhanced response against TAA noticed in this population. In perspective, therefore, the characterization of relevant immune parameters may also have a prognostic value, as recently emphasized by the finding that a decreased expression of immune response-associated genes was associated with poor prognosis, particularly in HER2+ cases [49].
Accordingly, the apparently preserved immunological capacity of HER2+ patients may constitute a favorable milieu for several immune system-based therapies. In this respect, a detailed characterization of the immunological profile at diagnosis may be useful to individualize the most promising therapeutic choice or to further contribute in the therapeutic schedule's design. Furthermore, since some chemotherapeutic drugs display beneficial effects on host immune functions [11, 50], our results suggest that a careful immune-monitoring of breast cancer patients during NC may be useful to predict the response to therapy and to obtain a better prognostic definition.
In conclusion, our data indicate that, compared with HER2- cases, patients with HER2-overexpressing, locally advanced breast cancer show a more limited tumor-related immune suppression. This may account for the clinical benefit achieved in this subset of patients with the use of drugs acting through immune-mediated mechanisms. These findings also provide the rationale for further studies aimed at assessing the possible predictive and/or prognostic role of immune markers in locally advanced breast cancer patients undergoing NC.
antibody-dependent cell cytotoxicity
- APCs:
antigen presenting cells
chromogenic in situ hybridization
- CTLs:
cytotoxic T lymphocytes
enzyme-linked immunosorbent spot
estrogen receptor
fluorescence in situ hybridization
fluorescein isothiocyanate
human epidermal growth factor receptor-2
granulocyte macrophage colony-stimulating factor
hematoxylin and eosin
hormone receptor
lymphokine activated killer
- mam-A:
- muc-1:
mucin 1
neoadjuvant chemotherapy
natural killer cell
- PBMCs:
peripheral blood mononuclear cells
phosphate-buffered saline
- PgR:
progesterone receptor
- Tregs:
regulatory T cells
tumor-associated antigens
central memory T cells
effector memory T cells
- TTemra:
terminally differentiated T cells
- TGF-β1:
transforming growth factor-β1
- TNF-α:
tumor necrosis factor-α
- Trag-3:
taxol-resistance associated gene 3
upper limit of normal.
The authors thank Miss S. Colussi for editing the manuscript and S. Bergamin, for his technical support. The study was supported in part by grants from the Associazione Italiana per la Ricerca sul Cancro (contract 10301 to R.D.).
Authors' Affiliations
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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text: Seller: southernlighting (9,295) 99.9%, Location: The Villages, Florida, Ships to: Worldwide, Item: 311739765254 Vintage style Cast Brass Embossed China Cabinet or Bookcase or Desk Lid Door Pull. Measures 1&1/2 inches tall, and the cast brass embossed backplate is 1&7/8 inches wide Comes complete with the mounting nails Nice low price and low shipping cost Any further information needed please email. Buyer to pay shipping costs. We take paypal or email for other options. Never a Handling charge. Returns are accepted if item is misdescribed, we want happy customers. Thank you for your business!! Froo www.froo.com | Froo Cross Sell, Free Cross Sell, Cross promote, eBay Marketing, eBay listing Apps, eBay Apps, eBay Application Condition: No damage, Material: Brass


text:
Backstory: The guy quit in a Social Big Team match on H3 before the game had started - I hadn't touched the guy. He then proceeds to have a full on breakdown whilst writing me a mini-essay. I can only assume this is mistaken identity. [url]http://imgur.com/a/VvkLF[/url] [url]http://imgur.com/a/p5HV5[/url] Any idea what he's on about? Anybody know this guy? I want to know why he had a breakdown over XBL. PS. If mods want me to remove his GT, just drop me a message on here. [Edited on 10.04.2012 5:04 AM PDT]
[quote][b]Posted by:[/b] kciduoyreksewmai [url=http://www.youtube.com/watch?v=kSyvr_y1nRw&feature=player_detailpage#t=122s]remonded me of this[/url][/quote]that was clearly filler material
[quote][b]Posted by:[/b] Make117 I loved the "just wait till the next MLG tournament" part hahaha[/quote]
Never knew that it was possible to rustle your own jimmies.
Boy am I glad I stopped playing Xbox a few years ago.
[url=http://www.youtube.com/watch?v=kSyvr_y1nRw&feature=player_detailpage#t=122s]remonded me of this[/url]
Either he mistook you for someone else, or I don't know.
[quote][b]Posted by:[/b] CND AAA Beef And that's why I don't play Halo anymore.[/quote] Because of people or laggy connection?
Hehehehe, I love getting messages like these.
Oh look, cay pissed someone off on live instead of the flood. What's the big deal?
[quote][b]Posted by:[/b] Cay330 [quote][b]Posted by:[/b] petitminou Dear OP, don't being a hax0r[/quote] [url=http://www.bungie.net/Stats/Halo3/Default.aspx?player=smellyfir&sg=1]Dear Petit, I'm not one. Thanks, OP.[/url][/quote] Dear OP, all my sarcasm, you can have it Thanks, petitminou
[quote][b]Posted by:[/b] petitminou Dear OP, don't being a hax0r[/quote] [url=http://www.bungie.net/Stats/Halo3/Default.aspx?player=smellyfir&sg=1]Dear Petit, I'm not one. Thanks, OP.[/url] [Edited on 10.04.2012 12:11 PM PDT]
Silly Goose, he could have simplified that to 23/25. OT: No idea who he is. Also, it made me laugh.
Yes... use your aggressive feelings boy, let the hate flow through you!
Dear OP, don't being a hax0r
Basically, he's demanding you pay him because of his laggy connection that causes him to lose. He then goes on to insult you and threaten your life report him, then contact Microsoft's xbox division about this. This is ridiculous. Troll or not. Too far. Try xbox.com.
He sounds about 8.
Anyone else read it in the Goliath's voice? From Borderlands 2.
The butt-hurt is strong with this one.
And that's why I don't play Halo anymore.
[quote][b]Posted by:[/b] WyIdfyre Either trolling or just raging at someone because he's sore about losing.[/quote] Hmm... Seems as though he's a [i]sore loser[/i]!
Man that is some anger!
[quote][b]Posted by:[/b] SH4D0W0733 [quote][b]Posted by:[/b] AJF1177 [quote][b]Posted by:[/b] Cay330 [quote][b]Posted by:[/b] WyIdfyre Either trolling or just raging at someone because he's sore about losing.[/quote] He didn't lose though. He quit before the game even had started. I originally assumed he was a troll, but jeez look at his GT & look at how persistent he is, along with the typing on an Xbox being utter hell to do; no sane person would troll using that many characters over XBl messaging.[/quote]There's a keyboard attachment for the controller that makes typing alot easier.[/quote] or you can jsut plug a keyboard into the console.[/quote] Doesn't fit in with the timings, as earlier stated.