text:
Top Mat
Bottom Mat
8.00" x 8.00"
8.00" x 8.00"
Flight Canvas Print
by Barbara Berney
Flight by Barbara Berney - 8" x 8"
Glossy Canvas
Gallery Wrap Stretched Canvas Print - 1.5" Stretcher Bars - Mirrored Image Sides
Assembly Charge
Product Details
Flight canvas print by Barbara Berney. Bring your artwork to life with the texture and depth of a stretched canvas print. Your image gets printed onto one of our premium canvases and then stretched on a wooden frame of 1.5" x 1.5" stretcher bars (gallery wrap) or 5/8" x 5/8" stretcher bars (museum wrap). Your canvas print will be delivered to you "ready to hang" with pre-attached hanging wire, mounting hooks, and nails.
Design Details
Flight is an original digital collage created through a series of layers, using photographic, fractal and hand-drawn images with original textures... more
Ships Within
3 - 4 business days
Additional Products
Digital Art
Canvas Print
Framed Print
Art Print
Metal Print
Acrylic Print
Wood Print
Greeting Card
iPhone Case
Throw Pillow
Duvet Cover
Shower Curtain
Tote Bag
Round Beach Towel
Carry-all Pouch
Beach Towel
Weekender Tote Bag
Portable Battery Charger
Bath Towel
Coffee Mug
Yoga Mat
Spiral Notebook
Fleece Blanket
Canvas Print Tags
Digital Art Tags
Comments (3)
Terril Heilman
Outstanding colors and flow to this abstract!
Barbara Berney replied:
Thank you so much!
Carol Elsip
Beautiful artwork, Barbara, I love the ethereal quality...
Barbara Berney replied:
Thank you, Carol. I find creating these calms my restless spirit. :)
Artist's Description
Flight is an original digital collage created through a series of layers, using photographic, fractal and hand-drawn images with original textures and colors. The layers are manipulated individually to achieve the desired effect. No "artistic" filters were used in this work. Some pieces may have as many as one hundred layers.
About Barbara Berney
I have been some sort of artist since I could wield a crayon as a precocious tyke. I've worked for 50 years as a graphic and fine artist, but I fell in love with digital art when I had the first of three thumb joint replacement surgeries. With my hands in casts for three summers in a row, the urge for creative expression overpowered. I quickly discovered that I could push the mouse around and click without much pain, and digital collage emerged. I create art because doing so infuses my spirit with a powerful, elemental connection to the universe. Each piece is an intimate expression of my own vitality, of the thrill of living every day. Each work of art offers an exploration of endless possibilities, encompassing the depth of my...


text:
WEB Bonaire had their electrical people certified again in June/September
On the island of Bonaire a lot of things has changed over the last years. A new electricity production company was introduced on the island an WEB Bonaire itself also made major investments in their distribution network. The people of WEB Bonaire are already working with this certification system for almost 15 years and about every 3 years the company makes another step forward. Network performance is improving every time. WEB Bonaire has a modern scada system to supervise th

subset: commoncrawl


text:
Brand new 2room-apartment - Port of Monaco - Monaco
- 2 Rooms
- Total Area 50 sqm
- Asking price 3 200 000 €
- Garden view
Le Suffren Monaco offers both residential and commercial real estate. Property investors will find a selection of studio, 1-bed, 2-bed, 3-bed, and 4-bedroom apartments, offering something for everyone, whether you seek an elegant pied-a-terre or voluminous proportions for family living. Many of the apartments at Le Suffren Monaco boast elegant fixtures and fittings, in addition to spacious terraces. Amenities at Le Suffren Monaco include a dedicated concierge service and secure parking, with many apartments also benefitting from a handy cellar, offering the utmost convenience.
If no property matches your search criteria
Please contact us for a personalized search
Le Suffren Monaco is in the district of La Condamine, Monaco's vibrant business district, located to the south-west of the city state.
The highlights of La Condamine include the famed Port Hercules, home to the world's most elegant superyachts, as well as its elegant seafront promenade with open-air swimming pool.
La Condamine is also home to the principality's only farmers market at the Place d'Armes, which has been in operation since 1880. This traditional market offers a selection of locally grown fresh produce alongside traditional cuisine and is a vibrant destination where a slice of authentic Monegasque life can be enjoyed.
Once a year this district also bursts into life as the epicentre of the famed Monaco Grand Prix which starts and finishes in this exclusive district.
There is no question that owning an apartment in Monaco's La Condamine district puts you in the very heart of this vibrant and exclusive destination.
When you buy an apartment at Le Suffren Monaco you benefit from everything that the Principality of Monaco offers.
Residents of Monaco enjoy a lavish lifestyle and access to world-class offerings, including luxury resorts, Michelin-starred restaurants, decadent spas, and some of the finest events in the world.
Monaco residents also enjoy a favourable tax system, political and economic security, a world-class healthcare and education system, and a convenient location along the French Riviera.
Technologically advanced and forward-thinking, Monaco was the first country in the world to make 5G technology accessible to all its residents and is also well on the way to reaching its remit of being carbon neutral by 2050.
La Costa Properties Monaco offers a refined selection of apartments in La Condamine, including at Le Suffren Monaco. Browse our portfolio of La Condamine real estate for sale, or contact one of our multilingual agents and let us help you find your ideal Monaco property.


text:
1994 Acura Integra Coupe LS 1.8 from North America
The best thing I ever owned
Front brakes and tires, oil, and I just had to replace the battery. That's it. I put 12,000 miles on it in 6 months.
General Comments:
It's my first Acura. I bought it for one dollar, and I will not sell it. I am keeping it till it dies.
It handles great, and is very reliable.
Would you buy another car from this manufacturer? Yes
Review Date: 26th January, 2011


text:
MANILA – Philippine Army (PA) troops on Friday transported 70 camper mats donated by bedroom fixtures company Uratex Foam to medical professionals of the Lung Center of the Philippines in Quezon City.
Reservist 2nd Matteo Guidicelli, in coordination with the management of Uratex Foam, was assisted by the PA in transporting the mats using military trucks from the company's factory in Canumay, Valenzuela City to the hospital.
He was also aided by the PA Civil-Military Operations Regiment in the transportation of the mattresses.
Meanwhile, PA commander Lt. Gen. Gilbert Gapay lauded Uratex's generosity to front-liners working to contain the spread of the coronavirus disease (Covid-19).
"Amidst the fight against Covid-19, your efforts will make our health professionals more comfortable that will boost their physical stamina in serving the people infected by the virus," he added.
Medical workers are among those exempted from the Luzon-wide enhanced community quarantine. (PNA)


text:
Contents2Introduction3Theory part: The use of games4 Note-taking10Practical part : Grammar games:14Speed14Spot the differences15Tipycal questions16Achievements16Reported advioce17Picture the past18Impersonating members of a set18No backshift19Incomparable20One question behind20Sit down then 22Only if22Two-word verbs23The world of take25A dictionary game 26Eyes27Umbrella28Listening to time 29Guess my grammar30Puzzle stories30Word ordwer dictation31Grammar lessons taking notes:33Passive voice33Context and meaning34Subject matter note taking36Conclusion37References38
This course work presents two teaching methods widely approved in Oxfrord Universities: grammar and vocabulary games and the variations of taking notes during the lesson.
Both of methods are embodied in the theory and practical part. As a theory part I give research works of professional lavguage teachers who studied the methods they considered as useful and effective and put their opinion and reseach works on the press. Im very grateful to them for sharing their experiences with us. So this part of my work describes the method itself, gives tests proving its effectiveness and touches some problem spots of it. Next I offer practical part containing examples of taking these methods in the classroom.
None of these methods presented here is any brand new discovery for the language teacher. Every teacher used to practice them in his/her work, theres only a try to add something new to well known and allegedebly usual techiques (like note-taking), to study them deeper and show more interesting and useful side of them. In short words some suggestions to make them work better.
The reason Ive chosen this theme is the wish to know more about how to make the lesson more interesting and useful at the same time. Ive benefitted much by collectiong and studing all this material I present here and hope youll find this work worth reviewing.
The Use of Games
For Vocabulary Presentation and Revision
by Agnieszka Uberman
Vocabulary acquisition is increasingly viewed as crucial to language acquisition. However, there is much disagreement as to the effectiveness of different approaches for presenting vocabulary items. Moreover, learning vocabulary is often perceived as a tedious and laborious process.
In this article I would like to examine some traditional techniques and compare them with the use of language games for vocabulary presentation and revision, in order to determine whether they are more successful in presenting and revising vocabulary than other methods.
From my teaching experience I have noticed how enthusiastic students are about practising language by means of games. I believe games are not only fun but help students learn without a conscious analysis or understanding of the learning process while they acquire communicative competence as second language users.
Vocabulary teaching techniques
There are numerous techniques concerned with vocabulary presentation. However, there are a few things that have to be remembered irrespective of the way new lexical items are presented. If teachers want students to remember new vocabulary, it needs to be learnt in context, practised, and then revised to prevent students from forgetting. We can tell the same about grammar.Teachers must make sure students have understood the new words, which will be remembered better if introduced in a "memorable way". Bearing all this in mind, teachers have to remember to employ a variety of techniques for new vocabulary presentation and revision.
Gairns and Redman (1986) suggest the following types of vocabulary presentation techniques:
- Visual techniques. These pertain to visual memory, which is considered especially helpful with vocabulary retention. Learners remember better the material that has been presented by means of visual aids. Visual techniques lend themselves well to presenting concrete items of vocabulary-nouns; many are also helpful in conveying meanings of verbs and adjectives. They help students associate presented material in a meaningful way and incorporate it into their system of language values.
- Verbal explanation. This pertains to the use of illustrative situations, synonymy, opposites, scales (Gairns and Redman ), definition (Nation) and categories (Allen and Valette ).
- Use of dictionaries. Using a dictionary is another technique of finding out meanings of unfamiliar words and expressions. Students can make use of a variety of dictionaries: bilingual, monolingual, pictorial, thesauri, and the like. As French Allen perceives them, dictionaries are "passports to independence," and using them is one of the student-centered learning activities.
Using games
The advantages of using games. Many experienced textbook and methodology manuals writers have argued that games are not just time-filling activities but have a great educational value. W. R. Lee holds that most language games make learners use the language instead of thinking about learning the correct forms. He also says that games should be treated as central not peripheral to the foreign language teaching programme. A similar opinion is expressed by Richard-Amato, who believes games to be fun but warns against overlooking their pedagogical value, particularly in foreign language teaching. There are many advantages of using games. "Games can lower anxiety, thus making the acquisition of input more likely" (Richard-Amato). They are highly motivating and entertaining, and they can give shy students more opportunity to express their opinions and feelings (Hansen). They also enable learners to acquire new experiences within a foreign language which are not always possible during a typical lesson. Furthermore, to quote Richard-Amato, they, "add diversion to the regular classroom activities," break the ice, "[but also] they are used to introduce new ideas". In the easy, relaxed atmosphere which is created by using games, students remember things faster and better (Wierus and Wierus ). Further support comes from Zdybiewska, who believes games to be a good way of practising language, for they provide a model of what learners will use the language for in real life in the future.
Games encourage, entertain, teach, and promote fluency. If not for any of these reasons, they should be used just because they help students see beauty in a foreign language and not just problems .
Choosing appropriate games. There are many factors to consider while discussing games, one of which is appropriacy. Teachers should be very careful about choosing games if they want to make them profitable for the learning process. If games are to bring desired results, they must correspond to either the student's level, or age, or to the material that is to be introduced or practised. Not all games are appropriate for all students irrespective of their age. Different age groups require various topics, materials, and modes of games. For example, children benefit most from games which require moving around, imitating a model, competing between groups and the like. Furthermore, structural games that practise or reinforce a certain grammatical aspect of language have to relate to students' abilities and prior knowledge. Games become difficult when the task or the topic is unsuitable or outside the student'sexperience.
Another factor influencing the choice of a game is its length and the time necessary for its completion. Many games have a time limit, but according to Siek-Piskozub, the teacher can either allocate more or less time depending on the students' level, the number of people in a group, or the knowledge of the rules of a game etc.
When to use games. Games are often used as short warm-up activities or when there is some time left at the end of a lesson. Yet, as Lee observes, a game "should not be regarded as a marginal activity filling in odd moments when the teacher and class have nothing better to do". Games ought to be at the heart of teaching foreign languages. Rixon suggests that games be used at all stages of the lesson, provided that they are suitable and carefully chosen. At different stages of the lesson, the teacher's aims connected with a game may vary:
- Presentation. Provide a good model making its meaning clear;
- Controlled practise. Elicit good imitation of new language and appropriate responses;
- Communicative prastice. Give students a chance to use the language .
Games also lend themselves well to revision exercises helping learners recall material in a pleasant, entertaining way. All authors referred to in


text:
The Greater Des Moines Partnership's Directory includes contact information and maps for all Regional Chamber Members in Greater Des Moines (DSM) — more than 6,500 local businesses! To search by business category, select the links below the search bar. To search by business name, select "0–9" for business names that begin with a number or "A–Z" for businesses listed in alphabetical order.
Additionally, The Partnership relies on Investors to support its mission as the economic and community development organization serving Greater Des Moines (DSM).
For more information about The Partnership, its Investors, Affiliate Chambers of Commerce and Chamber Members, visit the following pages:
We serve clients using the power of natural healing. We are committed to helping people rejuvenate and heal their bodies through massage, applied kinesiology, and bodywork techniques.
12871 University Ave., Ste. 180
Clive, IA 50325
- Map
- (515) 499-0099
HW CBD is a locally owned family company. Its proprietors have been involved growing the industry for nearly a decade and helped pass the law allowing for the sale of legal CBD products in Iowa.
1821 22nd St., Ste. 102
West Des Moines, IA 50266
- Map
- (515) 223-2370
Self care items. Handmade bath and body. Online Retail.
Unpublished address
Urbandale, IA 50322
- Map
- (515) 585-0600


text:
Telofisa is 64 years old. She married at age 15 and her husband died from natural causes 25 years ago. They were blessed with nine children. Seven of them have died and two of her sons are still living. Her sons have no reliable source of income. Telofisa stays alone in a single room. Sadly, she was rejected and abandoned by her family, making her living situation more difficult. She can hardly engage in any work due to her age. Telofisa gets support from well wishing people. Telofisa used to work at a food vendor but was stopped because she could not perform to her expectation. Currently, Telofisa is diabetic and hypertensive and has no ability to get adequate treatment and medications for these conditions. Despite her difficult circumstances, she is still a strong believer in Christ and praises God for her life. She was very happy when she heard about some generous people willing to support her in her conditions. "May God give them more years to live," she said with all her heart.
Women's Rights Without Frontiers is committed to helping Ugandan women widows, to ease their suffering and give them dignity and new hope in the twilight season of their lives.
Please help us help these women!
With gratitude for your generosity,


text:
UAL Level 3 Extended Diploma In Performing & Production Arts
Course Code
2 Years
Entry Requirements
Four GCSEs at grade 4 or above, or a level 2 qualification in a relevant subject area passed with merit or distinction with maths and English at grade 9-4 (or Functional Skills level 2 or above).
You will also need to complete a practical audition, including a monologue and / or acting workshop.
Future Study and Careers
Progression to university, drama school, starting an apprenticeship or paid employment.
This two-year course is for you if you are passionate about acting and would want to explore building a career in the performing arts.
You will learn through practical acting and devising workshops, which will be underpinned by research, group discussion, reflective journals, video diaries and evaluations.
You will be assessed in three public performances across the year at external theatre venues. One of the three performances will be part of the National Theatre Connections Festival.
You will learn essential techniques such as stage and on camera acting, voice technique, movement, devising, scriptwriting and contextual studies.
Our courses are awarded by University of Arts London and as a Level 3 student, you will have the opportunity to apply for their Insight Programme in London over the holiday periods.
Hitchin Campus


text:
Peters' father called the police Sunday night when she did not come home from visiting her aunt's house, Chippewa Falls Police Department (CFPD) said in a news release Monday. Later that night, officers found a bicycle in a wooded area by a walking trail close to her aunt's house, CFPD said.
Around 9:00 a.m. Monday morning, her body was found in the woods near the walking trail, according to CFPD. The Chippewa County Coroner's Office confirmed the remains were Peters'.
Officers believe Peters was last seen by family members the night of her disappearance, according to the CFPD release. She was supposed to be headed home from her aunt's house when she disappeared, CFPD said.
Overnight, local authorities searched for Peters by going door-to-door and using K9 teams, the release said. They also used a drone to search the wooded area where her bike was found.
During a news conference Monday, CFPD Chief Matthew Kelm did not elaborate on how the girl was killed or what evidence was found at the scene.
Police are investigating Peters' death as a homicide and do not currently have anybody in custody, Kelm said, adding police have set up a tip line for members of the community.
Kelm warned there may still be a danger to the public as authorities do not have a suspect in custody.
"The simple fact is that we have not made an arrest in this case, so the suspect is still at large," he said. "So in that case, we just want to be extra vigilant and make sure that if there are any tips, anyone has any information, to go ahead and call that tip line."
Multiple agencies are assisting CFPD in their investigation including the Wisconsin Division of Criminal Investigation and the Wisconsin State Crime Lab, Kelm said.
Quoted from Various Sources
Published for: The Bloggers Briefing


text:
L shaped couche are all the rage these days and are popping up in family rooms, living rooms, and dining rooms across the country. Not only do they look great but they also provide comfortable seating for large groups and are ideal when you want to be able to chat with friends or watch movies together without feeling crowded.
If you're thinking about purchasing one of these fantastic pieces of furniture then we've written this guide to help you find the perfect L shaped couch for your home and budget.
Different Choices and What to Look For: With such a wide variety of options available, it can be hard to pick out exactly what you want in an L shaped couch. That's why we've done all of that research for you! Use our guide below to figure out exactly what will work best in your living room. We'll also tell you about how much space it takes up and where each kind comes from. Enjoy! 🙂
Couch Buying Guide For Different Spaces
Many living rooms have room for a sofa and a love seat, but not enough space for both. If you are in need of seating on either side of a fireplace or other focal point, then an L shaped couch will be much more functional than two separate pieces. Make sure you measure your desired seating area before purchasing an L-shaped sofa. That way you can make sure it is long enough to accommodate guests and large enough that you don't feel cramped when relaxing on it.
How To Choose The Right Type Of Fabric Or Cover For Your Couch
When you purchase a couch or sofa, you will want it to last a long time. You also want it to look good and be comfortable. The following tips can help you choose fabric or covers that will give you what you need. Follow these tips so that you will get years of pleasure from your purchases.
Tips On Choosing The Ideal Size Sofa For Your Home
The sofa's come in a variety of sizes, shapes, and styles. Many people don't know what size of sofa they should buy. If you are thinking about purchasing a new sofa, be sure you know what size would best fit your room. There are many other things that can affect your decision on choosing which size sofa is right for you. such as whether or not it is leather if it has storage space under it, and if it comes with pillows or not!
How To Pick The Ideal Shape Of A Sofa
When looking for a sofa or couch, it's worth taking a step back and considering what type of space you want to fill with a new piece of furniture. How are you going to be using it? What colors will work best? Will they match any other items that are already in that room? These considerations and more should go into picking out a new piece of furniture, and sofas come in many different shapes! This can help narrow down your choices quite a bit.
What Are The Benefits Of An Adjustable Sofa?
Although not as common as other styles of L shaped sofa, an adjustable one can come in handy in various ways. One example is that they are often able to convert into a bed, which can be useful if you have guests who need to stay over. On top of that, they are also more affordable than a majority of standard sofas. If you're interested in purchasing an adjustable sofa, here's what you should know before making a purchase:
List of 10 Best L Shaped Couch in 2022
HONBAY L Shaped Couch with Linen Fabric Sofa Best Price
Brand: HONBAY
Shape: L-Shape
Frame Material: Wood
Belffin Convertible Sectional Sofa with Chaise L Shaped Couch reviews Best Price
Brand: Belffin
Seating Capacity: 3.00
Shape: L-Shape
HONBAY Convertible Sectional L Shaped Couch Grey Sofa Best Price
Back Style: Cushion Back
Frame Material: Wood
Size: 106.3″48.8″34.2″
l shaped couch with Faux Leather Sectional Brown Sofa Best Price
Brand: HONBAY
Style: Modern
Shape: L-Shape
Modular Sectional Sofa with Reversible Chaise L Shaped Couch Best Price
Brand: Belffin
Seating Capacity: 4
Assembly Required: Yes
Casa Andrea Milano Modern Sofa L Shaped Couch Best Price
Brand: Casa Andrea Milano
Type: Sectional
Seating Capacity: 5
Reversible Sectional Sofa L Shaped Couch for Small Space Best Price
Brand: HONBAY
Style: Modern
Seating Capacity: 3.00
Convertible L Shaped Couch Chaise with Ottoman Best Price
Brand: HONBAY
Shape: L-shaped sofa with Ottoman
Arm Style: Square Arm
Pawnova Convertible Sectional Sofa L Shaped Couch Best Price
Brand: HONBAY
Style: Modern
Room Type: Bedroom, Living Room
Casa Andrea Milano 3 Piece Modern l Shaped Couch Best Price
Brand: Casa Andrea Milano
Style: Modern
Seating Capacity: 4
Top 10 L Shaped Couch in 2022: Reviews
1) HONBAY L Shaped Couch with Linen Fabric Sofa
If you're looking to add some contemporary style to your home, then you might want to consider adding an L-shaped couch to your living room. These couches provide several different seating options in one area, allowing more than one person to sit down and relax at the same time. They also look very stylish with their plush seating and sleek designs, making them the perfect addition to your home if you're looking for furniture that's both comfortable and stylish.
What is so good about the Honbay sofa
First of all, it is really affordable. It has a wide range of colors to choose from. Most importantly, it looks very stylish and modern. In fact, it will look great no matter which room you place it in. Another good thing about buying this couch is that you can sit on both sides of it (face to face). And finally, even though it's reasonably priced, doesn't mean that you have to compromise on quality. This is a high-quality sofa after all!
Key features
The HONBAY WL-301 is a great option for those who want to get more use out of their space. This sectional is convertible into a bed, so it can add extra seating or sleeping space if you're expecting guests at any point. The leather material makes it easy to clean, while its modern design gives it a sleek touch that's perfect for your home. Plus, your dogs will love curling up on one of its cushions!
Pros and cons
Once you've researched your market and written down your business idea, it's time to do a reality check. It is good to look at both the pros and cons of starting your own business. What are you going to be getting out of it? What sacrifices will you have to make? Are there any potential downsides or pitfalls that should be addressed? Going into business for yourself is a big step, so consider carefully what you stand to gain or lose before jumping in.
2) Belffin Convertible Sectional Sofa with Chaise L Shaped Couch
Is it time to replace your old sofa? If you don't have much space, and you can't afford a sectional sofa, then the Belffin Convertible Sectional Sofa with Chaise L Shaped Couch may be just what you're looking for. It's roomy enough to seat three people comfortably (or maybe even four on a really good day), and yet it folds away when you want to make room in your living room or bedroom.
Should you buy convertible furniture?
In recent years, convertible furniture has gained popularity. This type of furniture is ideal for people who have a smaller home but want to get as much out of it as possible. To those unfamiliar with convertible furniture, it might seem like a total waste of money and unnecessary. However, if you're open to giving convertible items a chance, you might be surprised at how useful they can be in your home.
Key features
Belffin has a reputation for high-quality, affordable furniture. They're leaders in office furniture – everything from ergonomic chairs to filing cabinets, as well as executive desks and conference tables. They also offer home decor items like accent tables and mirrors. With Belffin you can be sure that your piece will last a long time, making it worth every penny you spend on it.
Pros and cons
There's always good and bad when it comes to any product. Before you buy, you should always learn about both. That's why we've compiled a list of pros and cons for Belffin convertible sectional couch so that you can see both sides before making your final decision. We don't want you to regret your purchase later.
3) HONBAY Convertible Sectional L Shaped Couch Grey Sofa
If you are searching for the perfect furniture to revamp your living room, then look no further than the HONBAY Convertible Sectional L Shaped Couch Grey Sofa. This sofa contains everything you need to make a huge statement in your home and impress your guests. Whether you are looking to redecorate or simply need some extra seating, the HONBAY Convertible Sectional L-Shaped Couch Grey Sofa will surely provide an exceptional decorating experience that everyone will enjoy!
The Benefits Of Owning A Convertible Sectional
There are tons of great reasons to buy a convertible sofa. Here are a few of them they're very affordable, they're available in a variety of styles and colors, and they can be adjusted in many different ways. For example, you can use some sections as sofas while others serve as beds or even couches! When you own a convertible sectional, you get several pieces that you can use separately or together depending on your needs at any given time.
Key Features
Sectional sofas offer a unique space-saving, the comfort-enhancing solution to your home decor. They also come in a range of styles and price points, making them versatile enough to suit your tastes and needs. Read on to learn more about how sectionals can solve common living room problems and how to choose one that's right for you! A sofa is a big investment—and should be treated as such. When shopping for a new sofa, don't just go by looks alone; consider longevity, comfort, and ease of use when choosing between different types of furniture. While upholstered couches tend to be more durable than their slipcovered counterparts, some slipcovers are made from material that will last years with proper care.
Conclusion And Recommendations
In conclusion, there are plenty of great convertible sectional sofas available on Amazon. Be sure to do your research before buying a new couch or sectional because you need to make sure it will fit in your home and look as good as it is going to feel. Thanks for reading my HONBAY Convertible Sectional L Shaped Couch Grey Sofa reviews. I hope it helps you in deciding which one is right for you!
4) Reversible Sectional Sofa L-Shaped Couch for Small Space
Want an easy way to add extra seating and lounging space to your small living room or den? Opt for a reversible sectional sofa, which gives you the best of both worlds when it comes to shape and style. A reversible sectional sofa allows you to flip between two distinct looks, depending on your mood or whether you're entertaining guests at home or relaxing alone with a good book.
Smaller sizes that won't overwhelm the room
Since you're dealing with a smaller space, you want to be sure your couch isn't too large for it. If you have little ones running around, furniture pieces with sharp corners or sharp fabric may be dangerous to them. On that note, look into softer fabrics like leather and microfiber. Both are easy on little fingers and toes—plus they clean up easily.
Reversible section
sectional sofas are just what they sound like: one big piece of furniture that can be broken down into individual seats. If you have a small space and are in need of some extra seating, such as an apartment or dorm room, these are worth looking into. They look just like normal sofa sets but break down when not in use. Their biggest advantage is that they can provide you with more seating than an average couch set.
Pros and cons
Sectionals are great options for open spaces. Sectional sofas can be turned around to fill any empty corners in your room, they often offer more seating than regular sofas (and are cheaper than buying two separate pieces), and they're easier to move around. But sectionals aren't always as practical as they seem. Because of their size, you may have trouble finding a place to store them when they're not in use. Plus, there are some downsides to choosing a reversible sofa.
For example, many reversible designs don't have cushions on both sides—meaning you'll need extra pillows or Heated Blanket if you want to face it one way or another. If that's not an issue for you and you like what you see here, then check out our list of top picks below!
5) Modular Sectional Sofa with Reversible Chaise L Shaped Couch
When you are looking to purchase new furniture, it is always a good idea to choose something that will be long-lasting and serve its purpose well. If you live in an apartment or home with limited space, you want your furniture to look as beautiful as possible without taking up too much space. Modular Sectional Sofa with Reversible Chaise L Shaped Couch will allow you to do both of these things, giving you the living room that you've always wanted in your house or apartment.
How a modular sectional sofa can improve your living room
As much as we love a gorgeous pair of Nursery Rocking Chairs or a delightful dining table, there's no denying that our living rooms are where we spend most of our time when at home. Whether it's watching TV, socializing with friends and family, or curling up to read a book by yourself—when it comes to domestic comfort, nothing beats spending quality time in your living room.
Key features
Clean-looking, modern design that works well in almost any room. One of my favorite features of modular sofas is how well they work in homes with varying sizes of rooms. This one has a sleek look that works really well in both big and small spaces. I love having versatility in my furniture pieces since I spend a lot of time out of the house traveling, so I can't always rely on being able to sit comfortably in my home office. With these sections, I know I'll have more options to work from no matter where I am!
Pros and cons
Modular sectional sofas are a practical and versatile choice, which can be designed to meet a wide range of needs. Here are some important things to consider before you make your purchase: Modularity offers flexibility in terms of layout—you can arrange it in a variety of ways based on your needs and personal preferences, making it suitable for large spaces or rooms where you need extra seating.
6) Casa Andrea Milano Modern Sofa L Shaped Couch
The Casa Andrea Milano Sofa and Loveseat set has quickly become one of the most popular pieces of furniture on the market, despite only hitting the market in March 2022. With its sleek design and modern appeal, this stunning piece will be sure to make an impression on your guests while not taking up much space at all, making it perfect for almost any room in your home or office. Although the Casa Andrea Milano may appear simple on the surface, it's actually made from top-quality materials and features some surprisingly advanced technology under the hood that you'll want to check out!
What makes Casa Andrea Milano stand out?
When it comes to modern design, one can't ignore Italian masterpieces. The bold lines and heavy detail found in Casa Andrea Milano furniture sets it apart from its competitors; each piece is a stunning conversation starter that will take your living room from drab to fab. Each component of their sofas is made with high-quality materials such as polyurethane and treated leather, meaning they're built to last through years of wear and tear.
Dazzling, rich, and elegant all in one place
that's what you get with a sofa. If you want to surround yourself with style and sophistication, investing in a sofa is one of your best options. There are many companies that produce furniture nowadays, but only a few can promise elegance combined with great quality. That's why we strongly suggest you go for products from leading Italian company Casa Andrea Milano and their amazing sofas collection.
Pros and cons
While some may argue that price is not an issue when it comes to quality design, others are not as willing to break the bank for something that will likely be used less than a handful of times over its lifespan. Fortunately, one Italian design house is coming out with a solution for both types of people by creating top-of-the-line furnishings for every home at an affordable price.
7) l shaped couch with Faux Leather Sectional Brown Sofa
Whether you're redecorating your living room or furnishing your first apartment, having the right furniture is important to make it feel like home. A lot of people opt for sectionals when they want to add style and comfort to their home, but not all of them are created equal. So before you make that next big furniture purchase, learn more about the L-shaped couch with Faux Leather Sectional Brown Sofa. Here are some helpful tips on what to look for in this type of sofa and what you can expect from it over time.
What is faux leather?
The term faux leather refers to a material that is designed to resemble real leather. This material is not made from animal skin but is actually a synthetic blend of materials designed to mimic leather. The best thing about faux leather furniture is that it requires little maintenance and can be cleaned using regular household items like soapy water, window cleaners, or vinegar.
Reasons to choose faux leather sectional sofas
These days, many homeowners choose sectional sofas instead of a sofa and love seat combination. Many people who want to buy a new living room set feel torn between buying separate pieces of furniture or one big sectional. However, there are many advantages to purchasing an L-shaped couch with faux leather sofa reviews. Check out these four reasons why you might want to make that choice.
Pros & Cons of faux leather sofas
Every choice has advantages and disadvantages. Whether you're shopping for a faux leather sofa or something else, take time to consider your needs before making a purchase. Remember that no matter what type of furniture you choose, make sure it fits in your home and is within your budget.
8) Convertible L Shaped Couch Chaise with Ottoman
If you have been feeling trapped in the same old living room furniture and are ready to do something different with your space, consider the convertible L-shaped couch chaise with ottoman. Not only will this change the way you live in your living room, but it will change the way you view furniture altogether. With this great piece of furniture at your fingertips, you will be able to use it in multiple ways, helping your furnishings meet any and all of your needs, no matter what they may be.
General Information
Modern living rooms today have a lot of furniture that is multi-functional. The convertible chaise/loveseat couch is an example of one such piece of furniture. There are also reclining loveseats and sofas, which open up to become beds, and sleeper sofas, which double as couches during the day and fold out into beds at night. So you see there are many options available to make your space more versatile.
Design & Features
The convertible chaise section of our couch is very popular among customers because it comes with a fold-out leg rest (or ottoman) that allows for extra lounging space, making it so much more than just an ordinary couch. And even though many don't buy it for its design, we do have to admit that it looks and feels quite comfy. The chaise longue can be positioned as a L shape or a U shape you choose!
Wrap Up
The general consensus among couch chaise lovers is that it's one of those pieces of furniture you cannot pass up. Aside from its affordability, you can also transform it into a twin-sized bed whenever you have overnight guests, and so on. While some people think it's not exactly practical to have a convertible piece of furniture in your living room, others say that it looks great.
9) Pawnova Convertible Sectional Sofa L Shaped Couch
If you live in an area that has cold winters and hot summers, you know how difficult it can be to find the right furniture to survive your temperature swings. But thankfully, there's the Pawnova Convertible Sectional Sofa, which can withstand all four seasons without sacrificing comfort or style. From its easy-to-clean microfiber fabric to its heat-molded cushions, this sofa has every feature that makes it perfect for year-round use. See why so many people love it in this informative article!
Introducing the Pawnova Sectional
If you love versatility, comfort, and style, then you'll adore the Pawnova Convertible Sectional Sofa. Not only is it an L-shaped sectional that provides ample seating when needed, but also easily converts into a large bed when guests come over or when you just want to get some extra rest. It's available in different colors and fits any living room décor thanks to its sleek design. See what makes it so special by reading our review!
Benefits of Buying Leather Sofa
Leather has stood out as a higher-end option, and understandably so. It's stylish, durable, and easy to clean—what's not to love? However, leather furniture tends to be more expensive than fabric furniture. To help you make an informed decision about whether or not leather is worth it for your home, here are a few benefits of buying a leather sofa.
Pros and cons
The pros of buying a convertible sectional are that it can be easily rearranged. At its most compact, you can use it as a couch and push it flush against your wall. But when you need extra space, you can expand it into a recliner and turn any room into an entertainment area. The biggest downside is the price—you'll pay more than $1000 at some retailers.
10) Casa Andrea Milano 3 Piece Modern l Shaped Couch
The Casa Andrea Milano 3 Piece Modern L Shaped Couch is an elegant yet comfortable set of sofas that will transform your living room into the perfect place to relax and unwind with friends or family. Check out these Casa Andrea Milano 3 Piece Modern L Shaped Couch reviews from real customers who have purchased this beautiful piece of furniture.
About the couch
It's sleek, stylish, and modern. And it looks great in a room, no matter its size or design theme! And there's more to love about Casa Andrea Milano's 3-piece L-shaped couch set; these loveseats feature nailhead accents that make them appear more expensive than they are, while their microfiber upholstery is easy to clean and helps prevent messes from soaking through to your floors or carpeting.
Style & Design
The Casa Andrea Milano Three-Piece Sectional Sofa is a piece of furniture with minimalist lines and a sleek design. With an L-shaped configuration, it can accommodate three people at once, but also feel roomy when used by just one or two. Featuring a modular construction, it's both sturdy and comfortable. The sofa comes in two different sizes (the larger one is about $650 more expensive) and four different colors: black, brown, cream and grey.
Materials & Craftsmanship
Casa Andrea's modern pieces are made of MDF. MDF is a particleboard product comprised of wood chips and then glued together to form sheets. The surface is coated with a layer of synthetic resin and heat-cured at high temperatures to give it strength, durability, and flatness.
Pros and cons
Casper (referring to Casa Andrea Milano 3 Piece Modern L-Shaped Couch) has a number of pros and cons. Pros: The furniture is absolutely gorgeous, soft, cozy, and worth every penny! Cons: Absolutely none! This piece of furniture is a must-have for any home. Not only does it look good but it feels great too! Highly recommended!
The 6 Things You Need to Consider Before Choosing an L-Shaped Couch
The L-shaped couch that you choose plays an important role in your home's decor, style, and functionality, as it is one of the largest pieces of furniture that you'll likely have in your living room or family room. So before you make the decision to purchase an L-shaped couch, there are several factors that you should consider to help ensure that the purchase will be beneficial to you and your home. Here are the 6 things you need to consider before choosing an L-shaped couch.
How much space do I have?
Most sofas come in different lengths and widths, so it's important to measure your available space first. Longer couches will require more floor space and may not fit in your room if you have a limited amount of space. Make sure that a sofa will be comfortable for all members of your household by measuring each person's height when seated; the armrest should fall naturally at hip level or slightly above.
What sort of style am I going for?
In order to choose an L sofa for your home, you first need to figure out what sort of style it is that you're going for. Are you looking for a modern and minimalist aesthetic? Would traditional furnishings fit in with your current design? Or do you like retro furniture? This will help you make a decision on whether or not that sleek, stylish piece of furniture is right for your living room.
How many people will be using the couch?
If you only have one or two people using it, you won't need anything fancy. However, if there are going to be four or more of you using it at any given time, you may want to consider a larger option. This will allow more people to sit comfortably while watching TV or hanging out together on a rainy day. Check out companies like Crate and Barrel for some great deals!
Is it easy to move around?
Sure, you might be thinking it's not a big deal if your couch isn't moveable; it sits in one place, right? While that may be true, you have to think about how easy or difficult it will be for you and/or your friends/family members to move that couch if you want or need to rearrange things. It could prove difficult—particularly if there are stairs involved—if your furniture is too heavy or unwieldy.
How long do I plan on having it?
While some couches are built for permanence, others are purchased with a more temporary purpose in mind. If you plan on having your couch for more than four years, it's worth investing in something quality that can last. However, if you're buying a couch for your basement game room or for your kids, and will probably get rid of it when they leave home, you don't need anything too fancy.
How much does it cost?
An L-shaped sofa is a large purchase, so you'll want to have a good idea of what you can afford. An average cost for an L-shaped couch is about $1000, but some can go for over $5000. Take into account what other furniture and accessories you'll need if you don't already own them. For example, you might need a new coffee table or end tables as well.


text:
Ywain and Gaheris will be coming down today at 11 AM EST / 4 PM GMT to address some issues accessing some dungeon regions.
We expect to be back online within the next 3 hours.
Thank you for your understanding and appreciate your patience as we work to get Ywain and Gaheris back online.


text:
100 PCT 026: Blogging and Social Media with Catherine Christaki
Archived series ("Inactive feed" status)
When? This feed was archived on September 14, 2019 01:47 (). Last successful fetch was on October 19, 2016 14:08 ()
Why? Inactive feed status. Our servers were unable to retrieve a valid podcast feed for a sustained period.
What now? You might be able to find a more up-to-date version using the search function. This series will no longer be checked for updates. If you believe this to be in error, please check if the publisher's feed link below is valid and contact support to request the feed be restored or if you have any other concerns about this.
Manage episode 163555207 series 1286107
Catherine is an industry expert. When it comes to combining Social Media, Blogging and Translating, she is right up there at the top of the tree.
So, as you can imagine, I was very pleased when Catherine agreed to come on the show and share some of her expertise. She doesn't disappoint: this show is packed full of great information for those interested in blogging, twitter, linkedin and the best way for translators to take advantage of the new technology that's out there. But perhaps more than that, this episode is about Catherine's story, how she started out and how she got to where she is today – from small beginnings to a very successful company with clients all around the world, and from Athens to Toronto in the process!
Thanks Catherine, it was great to have you on the show!
Here are Catherine's Contact details:
Twitter: @linguagreca Email: email@example.com
Also, at the beginning of the show, I mentioned the special limited-time offer for podcast listeners.
If you are interested in getting "How to Make More More as a Translator" at this very special price,
Click here to get your copy today!
All the best!
The post 100 PCT 026: Blogging and Social Media with Catherine Christaki appeared first on 100 Percent Translations.
37 episodes


text:
Skinny Pants
Discover Pinterest's 10 best ideas and inspiration for Skinny Pants. Get inspired and try out new things.
Shop JCrew.com for the Highest Quality Women's and Men's Clothing and see the entire selection of Children's Clothing, Cashmere Sweaters, Women's Dresses and Shoes, Men's Suits, Jackets, Accessories and more.
Sourabha Sn saved to Look !
Shop Fashion Nova super cute bottoms and find the pair that's right for you! With our wide selection of fashion bottoms, you're sure to leave happy!
Dashauney Lewis saved to Fashion
Material: CottonLength: Full LengthModel Number: 8175Waist Type: HighClosure Type: Button FlyStyle: Office LadyItem Type: JeansFabric Type: SoftenerJeans Style: Pencil PantsFit Type: skinnyWash: Snow WashWash: Bleach WashGender: WomenDecoration: Fake ZippersDecoration: PocketsDecoration: BowDecoration: Button
Omid Jadgan saved to My saves
it-girl - vestido-poa - poa - verão - street-style
Steal The Look saved to Looks para Roubar Agora
Looking for ways to make business casual look hot? Here are our best tips and tricks for dressing both professional and sexy!
Lacey Hanemann saved to Work
Pantalones pitillo de cintura alta con diseño de cremallera Online. Discover hottest trend fashion at chicme.com
Carlos Fernando saved to leggins


text:
By Julia Karow
This article, originally published Sept. 21, has been updated from a previous version to include additional information from a company official.
Pacific Biosciences said this week that it plans to focus on cancer and infectious diseases as initial application areas for its single-molecule real-time sequencing platform, noting that these areas are best suited to take advantage of the system's long reads and short cycle time.
In preparation for the full commercial launch of its instrument during the second half of 2010, the company has begun to build a sales force, and to scale up in the areas of manufacturing and operations.
PacBio is also working on additional applications for its SMRT analysis platform, to be introduced over time, including methylation sequencing, direct RNA sequencing, and protein translation.
The firm further disclosed that it is collaborating with six early-access customers, including Monsanto and the Scripps Institute, on a variety of sequencing projects. It will soon finalize the list of institutions that will receive an early commercial version of its SMRT sequencer during the first half of next year, which will likely include Monsanto and large academic genome centers.
At the UBS Global Life Sciences conference in New York this week, PacBio CEO Hugh Martin said that the company plans to focus initially on sequencing applications in cancer and infectious disease research, where it sees the strongest differentiation from its competitors.
The platform's long reads — expected to be at least 1,500 bases at launch next year — are especially sought after by cancer researchers, he said, and its short run time, or cycle time, of 15 minutes makes it attractive for infectious disease applications that require a fast answer.
At the moment, Martin said, the company has six collaborations underway that cover a variety of sequencing projects, including candidate variant discovery, agricultural biology, medical sequencing for cancer, and infectious diseases. Collaborators include three genome centers, two academic centers, and one company, which he later identified as Monsanto, an investor in PacBio's last funding round.
Also among the collaborators is Nicholas Schork's team at the Scripps Institute in San Diego, which uses PacBio data to study drug-resistant bacteria, according to PacBio Vice President of Marketing Martha Trela.
During the first half of next year, the company plans to ship on the order of 10 instruments — an early version of the commercial platform — to a number of institutions. Trela told In Sequence last week that the firm plans to "finalize the list" of these customers within the next couple of months. Monsanto is "very interested in being a customer," she said, and the firm "will be in discussions with big genome centers about their level of interest and criteria for those test sites."
In terms of customer types, Martin said the company will first concentrate on large genome centers and academic institutions, followed by pharmaceutical companies and "smaller individual customers," and finally on clinical diagnostics customers.
The company has already helped researchers with 45 grant applications that mention PacBio's platform "as the preferred piece of hardware," he said, and the firm believes it will be able to benefit from stimulus funding.
However, Martin cautioned that there are "only so many instruments" PacBio can manufacture next year and in 2011, and said that it will need to "think about customer diversity" so not all instruments go to one type of customer, such as genome centers.
[ pagebreak ]
He said the firm sees "quite a big opportunity" in the biotech and pharma market as well as the applied and industrial market, where traditional Sanger sequencing still dominates today.
According to Martin's forecast, the DNA sequencing market will grow from approximately $1.4 billion today, more or less evenly split between Sanger and next-gen sequencing, to about $1.8 billion by 2013, with the growth coming from next-gen sequencing.
Facing the Competition
Martin also squarely addressed the competition, and where the company believes it has an edge.
Roche/454's Genome Sequencer FLX will be "the product most directly impacted by us," he predicted. Users of the 454 platform have been mainly interested in its long reads, compared to other next-generation sequencing platforms, he said, and PacBio will be able to provide even longer reads at a lower cost.
Compared to Illumina's and Life Technologies' short-read platforms, which he said have "roughly the same kind of performance profile," even though Illumina's Genome Analyzer appears to be dominating in the market, PacBio will offer a new type of sequence data that will be fundamentally different from either of the two. After PacBio's platform launches, he predicted, there will likely only be room and sufficient "academic drive" to support one other next-gen sequencing platform.
Martin also addressed common criticism of its platform by others. Existing next-gen sequencing vendors say, for instance, that PacBio's accuracy is inferior to that of their respective platforms, but he said that is only true for raw read accuracy, not for accuracy of "finished" sequence. He said that over the next two months or so, the company will start an "education process," through a series of papers, to inform researchers about the nature of its data.
He also addressed claims that PacBio's throughput will never be as high as that of second-generation sequencing systems because of the optical requirements for real-time sequencing. While that is true in general, he said, people will be "very surprised" when the company reveals its initial throughput next year, which relies on recent "breakthroughs" in sensor technology that he said the company has heavily invested in.
The throughput of the first instrument version can be increased several-fold in two ways without hardware changes, he explained. First, the company could switch the current DNA polymerase, which runs at 3 to 5 bases per second, for one that works at 15 bases per second, he said, noting that PacBio is already running sequencing experiments internally at 10 bases per second. In addition, the company is working on ways to actively fill the zero-mode waveguides with polymerase, thus increasing their occupancy from currently one-third to 80 percent to 90 percent.
Long term, Martin said, the company wants to develop an upgraded instrument that can generate 100 gigabases of sequence per hour. Reads could grow to between 50,000 and 100,000 base pairs, and the company has already developed a polymerase that can synthesize DNA at 25 bases per second, which he said could be increased to 50 bases per second. The company is already working on a chip with a million zero-mode waveguides, which it plans to introduce in 2013 or 2014. Martin also mentioned that the company might not always use fluorescent labels but may instead switch to other types of labels in the future.
Although PacBio has not revealed all specs for its first commercial instrument, Martin said it will have reads of at least 1,500 bases and a "time to result," or run time, of 15 minutes. A run will cost approximately $60 in reagents, and an experiment — including sample preparation, which does not require amplification — can be completed in a single day, which no other next-generation sequencer except for 454's achieves today.
PacBio has said previously that the first commercial version of the instrument will cost as much as current high-throughput sequencers, or on the order of half a million dollars, though Martin noted this week that the company will not price the instrument "right on top" of Illumina's Genome Analyzer.
The company expects that users will purchase on the order of hundreds of thousands of dollars worth of consumables per year for the instrument, according to Trela, similar to what Illumina has said its GA customers spend. A single sequencing experiment will cost "no more than in the low hundreds," in total, according to Trela.
[ pagebreak ]
Asked what the biggest near-term hurdle for the company is, Martin said that the instrument is "a very complex piece of equipment" with more than 3,000 components, and the company is currently most focused on executing the assembly and quality-control of its instruments.
Looking Beyond DNA Sequencing
Long-term, the company is also banking on other applications for its platform besides straight DNA sequencing. Trela said that the firm knew from the start that its technology, the principles of which were first published by Cornell University researchers in 2003, was amenable to study different types of single-molecule interactions, but it decided to focus on DNA sequencing first.
Since then, "we have also had a lot of people come to us and say, 'I'd love to look at RNA directly, or at ribosomes, or other types of applications,'" she said. DNA methylation analysis, in particular, "has been one of the applications the scientific community has shown a tremendous amount of interest in, and we think it could add much value," she said.
Partly in response to these requests, the company decided to work on additional applications for its platform, both internally and through scientific collaborations. New applications include methylation sequencing, direct RNA sequencing, and the analysis of protein translation. For the latter — observing ribosomes as they translate messenger RNA into protein — the firm is collaborating with an undisclosed research group at Stanford University.
It is unclear yet whether any of the new applications will be available when the platform is first launched later next year for DNA sequencing. "We'll develop them as quickly as we can," Trela said.
At last week's Personal Genomes conference at Cold Spring Harbor Laboratory, PacBio Chief Technology Officer Steve Turner presented early results for DNA methylation and RNA sequencing (see related article in this issue). He said that direct RNA sequencing will be available within a year of the platform's launch.
In preparation for the full launch of the instrument in the second half of 2010, PacBio has also started to build out its commercial arm, scaled up manufacturing and operations, and begun to assemble a sales force. The firm just hired Pat Brooks, a former vice president of sales for Affymetrix, as vice president for North American sales. It also added Randy Livingston, chief financial officer of Stanford University, to its board of directors.
Deciding in June to build out its own commercial operations, Martin said, was "effectively a vote by the board to become an independent public company." He said PacBio has "a strong desire" to go public and will "think about an IPO" next year if the market conditions are right.
The company has approximately 290 employees, and "we are continuing to grow and add," Trela said, but did not put a specific number on how much the staff is expected to increase in the near term.
PacBio expects to be profitable by mid-2012, and to spend $350 million in total before it becomes cash-flow positive. To date, it has raised $266 million in venture capital.


text:
Drug Treatment Centers in Gunnison, CO
Midwestern Colorado Mental Health Ctr
Midwestern Colorado Mental Health Ctr is based in Gunnison, Colorado - (710 North Taylor Street 81230).
Midwestern Colorado Mental Health Ctr allows Cash Payment, Medicaid Insurance, Insurance Through Medicare for the various services that it provides, including Cognitive and Behavioral Therapy, Services to Reduce the Risk of Relapse. It provides its services in various settings, including Outpatient Drug Abuse Treatment.
The center offers all of their services for the following care types: Addiction Rehabilitation Services. Lastly, the center has a number of individualized programs for individuals, including Aftercare and Relapse Prevention, Substance Abuse Testing, Mental Health Disorder Treatment.
We can help you find the right treatment facility that best fits your overall needs and financial requirements.


text:
Ads: Hobbies, crafts
Category: All categories > Buy and Sell > Hobbies, crafts
Location: Canada > Kamloops [Change location]
Shown below are free ads available in Hobbies, crafts, Kamloops. You will find classifieds for sale and to buy. Please click a classified ad below for more details or to reply to it.
Results found: 1
28-10-2018 22:08Offering: Hobbies, crafts in Canada, Kamloops ... View detailed ...
Please join us Saturday November 3rd at Gateway City Church 163 Oriole Road from 10 am to 2 pm. Here you will find local Home Crafters, Businesses and Local Merchants coming together and supporting the community. All funds going towards BC Children's Hospital. Our major Sponsor Save On Foods will be there with raffle prizes galore.
Results found: 1
Canada free classified ads for Hobbies, crafts, Kamloops, Hobbies, crafts free ads Kamloops, Hobbies, crafts for sale Kamloops, Hobbies, crafts to buy in Kamloops.
Our button:
Button code


text:
Kailee McKenzie
Most Popular
YouTube beauty guru and vlogger whose content relates to fashion, beauty, dance and lifestyle. She also has a popular vlog channel called Daily Kailee which features bloopers, outtakes and challenge videos.
She launched her main, beauty YouTube channel on June 30, 2009. Her vlog channel was previously called LittleMissKailee.
She moved to New York City to study fashion. Her main channel has more than 310,000 subscribers. She partnered with Style Haul.
She is from West Virginia and has a younger sister name Tori who has been featured in several videos on Kailee's channel, including "Tori's Makeup Look" and "Tori's Makeup Tutorial."
She and Emily June have collaborated on YouTube.


text:
Wild Flower Honey – 400mg THC – CBD – Sweet Jane
Wild Flower Honey – 400mg – Sweet Jane
Pure and unpasteurized honey from the valleys of Beautiful British Columbia. Medium Amber in Colour with a rich flavour. Only the freshest ingredients with no additives, preservatives, or artificial colouring, combined with 400mg of high-quality THC oil and CBD oil ( 50mg/ per tablespoon), our Wild flower Honey has a lush floral undertone that brings out the best of both the honey and the therapeutic properties of THC or CBD.
Comes in CBD or THC
400mg THC, 400mg CBD
There are no reviews yet.
You must be logged in to post a review.


text:
38 Happy Memories That Only 90s Kids Will Feel Nostalgic About
We all know that school life was the golden period of our lives but gone are the days for every 90s kid. We often say we don't realise the value of the moment till it's gone, the same goes with school memories.
Also, 90s school kids were probably the last generation that spent time outside playing with friends and not on mobile games. 90s kids have school friends, not Facebook friends. We were the last generation of kids who truly knew how to be kids. These days, playing outside has been replaced by sitting inside texting, playing video games and surfing the internet.
All of us have great memories from the 90s school life as opposed to social media life and digital networking. So, lets relive some beautiful memories of school life during the 90s.
1. Teacher teaching and legends eating.
2. Only legend said the blue part could erase ink.
2. Choosing the best name slip is the most challenging task.
3. When parents say study and the only thing that comes to mind is drawing and colours.
4. When you don't bunk classes to play cricket on the field but play off-field. Book cricket!
5. When the teacher is absent from class.
6. Best first benchers game for 90s kids.
7. When you forget your homework.
8. Only for backbenchers.
9. When the history syllabus is over and civics began.
10. Best carbon copy.
11. Ever put a proxy on the attendance register?
12. Mimicking your teacher after class.
13. Exam pad was occasionally the cricket bat.
14. Last page of notebook was a like play ground for indoor games.
15. You remember this key?
16. Backbenchers still don't know how to use divider in geometry.
17. When the light goes out in tuition class.
18. Free time = fun time.
19. Legends said if you keep a peacock feather in your notebook, it will give birth to many more feathers!
20. Today's kids will never know the choices you had to make.
21. Best way to block incoming calls when teacher threatened to call your parents.
22. Today's kids will never know the struggle when you lose one of the pieces of this pencil.
23. If you didn't play this game ever you missed 90s.
24. After attending math class pen be like.
25. When you think about the boring lecture.
26. When you try to understand mathematics.
27. When you attend history class.
28. Colour, colour which colour do you choose?
29. Old is gold.
30. The best way to begin the school year in 90s.
31. The right time to take revenge your 'friends'.
32. Jaane nahin denge tujhe.
33. Difference between backbenchers and frontbenchers when teacher punishes.
34. Million Dollar moment when you got stars in your notebook.
35. Breakfast of 90s legends.
36. Most popular drawing of 90s school kids.
37. Only legends say sharpen the pencil and put the waste in hot water, and it will become eraser.
38. Everyone waited for this moment.
The Conversation (0)
Start a conversation, not a fire. Post with kindness.


text:
Oligonucleotides containing unmethylated CpG motifs (cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN)) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. We evaluated the cellular mechanisms responsible for this effect. Development of a CTL response was enhanced when mice were immunized with peptide-pulsed dendritic cells (DCs) treated with CpG ODN. However, in vitro, CpG ODN had no direct effect on highly purified T cells. In vitro, CpG ODN treatment of peptide- or protein-pulsed DCs enhanced the ability of the DCs to activate class I-restricted T cells. The presence of helper T cells enhanced this effect, indicating that treatment with CpG ODN does not obviate the role of T cell help. The enhanced ability of CpG ODN-treated DCs to activate T cells was present but blunted when DCs derived from IL-12 knockout mice were used. Fixation of Ag-pulsed, CpG ODN-treated DCs limited their ability to activate T cells. In contrast, fixation had little effect on DC activation of T cells when DCs were not exposed to CpG ODN. This indicates that production of soluble factors by DCs stimulated with CpG ODN plays a particularly important role in their ability to activate class I-restricted T cells. We conclude that CpG ODN enhances the development of a cellular immune response by stimulating APCs such as DCs, to produce IL-12 and other soluble factors.
Oligodeoxynucleotides containing unmethylated CpG motifs (cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN))4 can activate B cells, monocytes, and NK cells and induce production of a wide variety of cytokines (IL-6, IL-10, IL-12, IL-18, IFN-α, IFN-γ, and TNF-α) (1, 2, 3, 4, 5, 6, 7). CpG ODN can also function as potent immune adjuvants (8). In a number of murine systems, CpG ODN enhances the Ag-specific Ab response, shifting it toward Abs of the IgG2a isotype (indicating a Th1 response) and induces development of enhanced CTL activity (8, 9, 10, 11, 12, 13).
Dendritic cells (DCs) are potent APCs and are creating considerable excitement in their ability to induce a cellular immune response (reviewed in Refs. 14, 15, 16, 17). Studies in animal models demonstrate that pulsing DCs with Ag can result in Ag-specific immune responses (18, 19, 20). Recent clinical trials suggest DC therapy may be effective in humans as well (15, 21, 22).
Extensive evidence indicates that the ability of DCs to induce a CTL response is dependent on T cell help (23, 24, 25, 26). In fact, there is extensive cross-talk between CD4+ T cells and DCs. Presentation of class II-restricted epitopes by DCs to CD4+ T cells results in activation of the CD4+ cells (24). CD4+ T cells also express CD40 ligand, and further activate DCs by signaling via CD40. This, in turn, enhances the ability of the DC to activate class I-restricted T cells. This effect is particularly important for naive T cells. Indeed, activation of DCs by CD40 ligand enhances the ability of the DCs to prime CTLs in the absence of CD4+ cells (27, 28, 29).
CpG ODN have been shown to be able to activate DCs in both the human and murine systems as indicated by induction of cytokine production, including IL-12 as well as other soluble factors, and up-regulation of class I MHC, class II MHC, CD80, and CD86 (30, 31, 32). In addition, there are reports of possible direct costimulatory effects of CpG ODN on T cells (2, 9, 33). However, little is known about which of these changes contributes most significantly to the development of an enhanced cellular response. We therefore evaluated how treatment of DCs with CpG ODN impacts on development of an enhanced class I-restricted T cell response.
Materials and Methods
Mice and cell lines
C57BL/6 and C3H female mice were purchased from Harlan Sprague Dawley (Indianapolis, IN). All mice were housed at the animal care unit of the University of Iowa and used at 8–10 wk of age. Naive C57BL/6 OT-1 mice that are transgenic for the TCR which recognizes MHC class I H-2Kb-restricted OVA 257–264 (SIINFEKL) in H-2Kb (34) were obtained from Tim Ratliff (University of Iowa). C57BL/6-IL-12 knockout mice (35, 36, 37, 38) were obtained from The Jackson Laboratory (Bar Harbor, ME). In CTL assays, EL4 cells (murine T cell lymphoma that is H-2Kb restricted and thus C57BL/6 mouse syngeneic) and/or EG7 cells (EL4 cells which contain a plasmid which expresses OVA) were used as targets (39). In select experiments, C57BL/6 mice were infected with an adenoviral vector that expressed OVA. Mice were injected i.p. with 1 × 108 PFU/mouse with the adenovirus designated Ad5TrF-OVA (5 × 1010 PFU/ml or 1.2 × 1012 particles/ml) generated by the University of Iowa Virus Core Lab.
In vitro generation of DCs
DCs were generated using a modification of a previously described approach (40). Bone marrow cells were obtained by flushing the pelvis, femurs, and tibias of C57BL/6 wild-type or IL-12 knockout mice. RBCs were lysed using 0.83% ammonium chloride solution. T cells were removed by complement-mediated lysis using a mixture of anti-CD3 (145.2C11), anti-CD4 (GK1.5), and anti-CD8 (53.6.7) Abs. B cells were removed by panning using a flask coated with anti-B220 (6B2) Ab. Remaining cells were allowed to adhere overnight. Nonadherent cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 50 μM 2-ME, 10 U/ml penicillin, 10 μg/ml glutamine supplemented with 1000 U/ml murine GM-CSF (R&D Systems, Minneapolis, MN), and 1000 U/ml murine IL-4 (PeproTech, Rocky Hill, NJ). Media was changed at 4 or 5 days, and DCs were harvested and used on day 7. DC phenotype and morphology was confirmed by flow cytometry and scanning electron microscopy.
CpG ODN treatment and Ag pulsing of DCs
A panel of CpG ODN were purchased commercially (Operon Technologies, Alemeda, CA). All ODN had a nuclease-resistant phosphorothioate backbone and were confirmed to have undetectable endotoxin content before use. The immunostimulatory CpG ODN designated 1826 (5′-TCCATGACGTTCCTGACGTT-3′), which has proven to be an effective adjuvant in our previous studies (13), was used as the immunostimulatory agent. CpG ODN 1982, a non-CpG-containing ODN (5′-TCCAGGAGTTCTCTCAGGTT-3′), and CpG ODN 1845, which is identical to 1826 except that the cytosine residues are methylated, were used as controls. Freshly prepared DCs were treated with 5–6 μg/ml CpG ODN for 12–48 h, washed, and then pulsed with 100 μg/ml to 5 mg/ml OVA (Sigma, St. Louis, MO), 2–200 μg/ml SIINFEKL peptide (Research Genetics, Huntsville, AL), or unsupplemented media for 12–24 h and washed twice before use.
Generation and measurement of CTL activity
DCs (5 × 104–1 × 106) generated and treated as above were injected i.p. into C57BL/6 mice. After ∼9 days, spleens from immunized mice were harvested, homogenized, and RBCs lysed with 0.83% ammonium chloride solution. Splenocytes from naive C57BL/6 mice were homogenized, RBCs lysed, incubated with 100 μg/ml SIINFEKL for 3 h at 37°C, and irradiated with 3000 rad. These peptide-pulsed, irradiated splenocytes (2 × 106/ml) were then washed twice and mixed with splenocytes (2 × 106/ml) from immunized mice in a 1:1 ratio in 10 ml 10% culture medium (CM) in a 25-cm2 flask and incubated at 37°C for 5 days. After the 5-day restimulation, lymphocytes (effectors) were obtained by centrifugation over Histopaque 1083 (Sigma). Effector cells were then incubated at various E:T ratios with 51Cr-labeled target cells. Target cells included EL4 cells (Ag negative), EL4 cells incubated with 100 μg/ml SIINFEKL for 1 h at 37°C and washed twice, or EG7 cells. Supernatant was harvested after 6 h and release counts were determined using a Beckman gamma 5500 counter (Beckman Coulter, Fullerton, CA). All samples were run in triplicate. Percent specific lysis was determined according to the following equation: (experimental cpm − minimum cpm)/(maximum cpm − minimum cpm) × 100.
Phenotypic analysis
DCs were stained with FITC-conjugated anti-MHC class I (anti-H2Kb), anti-B7-1 (1G10), anti-B7-2 (GL-1), and anti-SIINFEKL-H2Kb (25-D1.16) (41) using standard techniques. T cells were stained with anti-CD3 (145.2C11), anti-CD4 biotin (GK1.5), anti-CD8 biotin (53.6.7), and anti-B220 biotin (6B2). Propidium iodide staining was used to exclude dead cells. Stained cells were analyzed using a FACScan flow cytometer (Becton Dickinson, Mansfield, MA). Appropriate isotype controls were used.
Cytokine analysis
CpG ODN treatment of purified T cells
Spleens were harvested from C57BL/6 or C3H mice and homogenized, and the lymphocytes were separated by centrifugation over Histopaque 1083 (Sigma). These lymphocytes were panned for 1 h at 4°C, first with a mixture of Abs against B cells, MHC class II, and heat-stable Ag and then with a mixture against Fc receptors, erythroid cells, and B cells (courtesy of Tom Waldschmidt, University of Iowa). The resulting T cells were labeled with anti-CD5 (positive staining) and 6B2 (negative staining) for sorting on an Epics IV flow cytometer (Coulter, Miami, FL). T cells were >99.5% pure as demonstrated by FACS. One hundred thousand T cells were incubated with 5 μg/ml CpG ODN or control ODN. In select samples, 10 μg/ml anti-CD3 or 100,000 unfractionated irradiated splenocytes were added. After 48 h, supernatant was harvested and assayed for the presence of IL-2 using an ELISA per the manufacturer's protocol (Endogen, Cambridge, MA). Proliferation was determined by scintillation counting after labeling for 6 h with 1 μCi [3H]thymidine (Amersham, Arlington Heights, IL). All samples were tested in triplicate.
In vitro Ag presentation by DCs and T cell activation
Splenocytes from C57BL/6 wild-type, TCR-transgenic OT-1 mice (34), or mice infected with an adenovirus that expresses OVA were finely minced and RBCs were lysed using 0.83% ammonium chloride solution. Adherent cells were removed by plastic adherence. B cells were depleted by panning with anti-B220 (6B2) Ab-coated flasks.
In select experiments, purified CD8+ and CD4+ T cells were obtained by magnetic bead separation of T cells using either anti-CD8 (53.6.7) or anti-CD4 (GK1.5) magnetic beads according to the manufacturer's specifications (MiniMACS; Miltenyi Biotec, Auburn, CA). All samples were passed over the column twice and were 99% pure as determined by flow cytometry. In experiments where indicated, anti-CD40 Ab (kindly provided by Gail Bishop, University of Iowa) at 10 μg/ml was used in lieu of CD4+ T cells.
DCs generated and treated as above were added to T cells for 48 h at a T cell:DC ratio of 100:1. For proliferation assays, 1 μCi/well [3H]thymidine (Amersham) was added 6 h before harvest. To confirm that thymidine uptake was not due to DCs, control wells containing DCs alone were included. DCs alone demonstrated no significant thymidine uptake. Parallel samples were run without the addition of [3H]thymidine, and supernatant was harvested for cytokine analysis. All tests were performed in triplicate.
CpG ODN enhances the ability of peptide-pulsed DCs to induce a CTL response
Before exploring the cellular mechanisms involved, it was important to confirm that treatment of DCs with CpG ODN enhances the immune response in the system under investigation. Therefore, initial studies evaluated whether CpG ODN enhances development of a CTL response following immunization with DCs in vivo. C57BL/6 mice were immunized with DCs incubated with 10% CM, CpG ODN, or CpG ODN and SIINFEKL. After 9 days, spleens were harvested and incubated with irradiated naive splenocytes that had been incubated with SIINFEKL. After 5 days, live cells were harvested and the standard CTL assay was performed with targets as indicated. As illustrated in Fig. 1, CpG ODN significantly enhanced the ability of Ag-pulsed DCs to induce a CTL response against either EL4 cells incubated with peptide (Fig. 1,A) or against EG7 cells that also express target peptide (Fig. 1 B). Preincubation of DCs with CpG ODN before injection was needed to induce the enhanced CTL response. When DCs were pulsed with peptide, then coinjected in vivo with CpG ODN, no enhanced CTL were observed (data not shown).
CpG ODN has no detectable direct effect on T cells in the absence of APCs
As outlined above, previous reports suggest that CpG ODN may contribute to T cell activation by supplying a costimulatory signal directly to the T cells in the absence of other cell types. To assess this further, CpG ODN was added to sort-purified naive T cells in the presence or absence of CD3 cross-linking. Proliferation of T cells was then determined. As illustrated in Fig. 2, CpG ODN had no specific effect on T cell proliferation with or without CD3 cross-linking. Both CpG and non-CpG ODN had modest, nonspecific effects on the proliferation of T cells after CD3 cross-linking. Proliferation of T cells was seen in the positive control when anti-CD3 and APCs in the form of radiated splenocytes were added. Similar results were found using IL-2 production to detect T cell activation or CD3-based bispecific Abs to activate T cells (data not shown) (43). These data indicate that CpG ODN do not have direct effects on T cell activation in the presence or absence of CD3 cross-linking.
CpG ODN enhances the ability of peptide-pulsed DCs to activate MHC class I-restricted T cells
We next evaluated whether treatment of DCs with CpG ODN enhances their ability to activate class I-restricted T cells. DCs were treated with CpG ODN and pulsed with SIINFEKL peptide. Unfractionated T cells from naive C57BL/6 OT-1 mice, which are transgenic for a TCR that recognizes OVA254–263 peptide (SIINFEKL) in H-2Kb, were incubated with DCs obtained from wild-type C57BL/6 mice. T cell proliferation was determined using a standard [3H]thymidine assay and IFN-γ production as determined by ELISA. As illustrated in Fig. 3, A and B, peptide pulsed-DC induced both T cell proliferation and IFN-γ secretion. This effect was enhanced significantly by treatment of DCs with CpG ODN. No effect was seen when control, nonstimulatory, ODN were used (data not shown). CpG ODN had no effect on DC-induced proliferation of T cells in the absence of peptide, demonstrating that the effect of CpG ODN was Ag specific. This response was dose dependent, with increased T cell activation occurring with increased doses of peptide (Fig. 3 C). These studies confirm that CpG ODN enhances the ability of DCs expressing the target peptide to activate class I-restricted T cells.
CpG ODN enhances the ability of DCs pulsed with soluble protein Ag to activate MHC class I-restricted T cells
We next sought to determine whether CpG ODN impacts on the ability of DCs pulsed with soluble protein Ag to induce activation of class I-restricted T cells. DCs were treated with CpG ODN as outlined above and pulsed with soluble OVA. Naive OT-1 T cells were then added, and T cell proliferation and activation were measured as outlined above. As illustrated in Fig. 4, CpG ODN enhanced the ability of OVA-pulsed DCs to induce proliferation (Fig. 4,A) as well as production of IFN-γ secretion (Fig. 4 B) by class I-restricted T cells. No effect was seen with control ODN (data not shown).
The presence of T cell help enhances activation of class I-restricted T cells by DCs
The studies illustrated in Figs. 3 and 4 involved unfractionated T cells from OT-1 mice. These included CD4+ T cells, which could have supplied T cell help. We therefore used magnetically purified CD4+ and CD8+ T cells to assess the role of T cell help in the activation of MHC class I-restricted T cells by CpG ODN-treated DCs. CpG ODN-treated DCs pulsed with either protein (Fig. 5,A) or peptide (Fig. 5, B and C) induced a low level of activation of purified class I-restricted CD8+ T cells in an Ag-restricted manner in the absence of T cell help. The addition of T cell help significantly enhanced activation. Fig. 5,B (measuring IFN-γ production) and Fig. 5,C (measuring proliferation) show similar findings using the MHC class I immunodominant epitope of OVA (SIINFEKL) as the Ag. Non-CpG ODN controls, not shown in Fig. 5, B and C, revealed results similar to those in Fig. 5 A. T cell help could be supplied by either Ag-nonspecific T cells (wild-type CD4 T cells), CD40 ligation via anti-CD40 Ab, or by Ag-specific T cells (T cells obtained from mice infected with an adenovirus that expressed OVA referred to as Ad-OVA CD4 T cells). Specific T cell help was most effective at enhancing CD8+ activation. Overall, these results demonstrate that CpG ODN treatment of DCs can enhance the ability of DCs to activate CD8+ T cells in the absence of T cell help. However, T cell help further enhances this activation. Although help can be Ag nonspecific in this system, the CD8+ T cell response is enhanced most extensively when specific T cell help is supplied.
Cytokine production by CpG ODN-treated DCs is responsible for the enhanced class I-restricted T cell response
Previous studies demonstrated that CpG ODN can enhance MHC expression, increase expression of costimulatory molecules, and enhance production of soluble factors by DCs and other APCs (6, 32). We explored the relative importance of these changes in the ability of CpG ODN-treated DCs to induce activation of class I-restricted T cells. In initial studies, we compared the ability of fixed and viable peptide-pulsed DCs to activate T cells. CpG ODN treatment of DCs had no significant effect on the amount of peptide presented in class I as demonstrated by flow cytometry using an Ab specific for the SIINFEKL-MHC I complex, and fixation did not alter expression of this complex (data not shown). However, fixation of DCs after exposure to CpG ODN eliminated their ability to enhance IFN-γ secretion by class I-restricted T cells (Fig. 6). Thus, fixation of peptide-pulsed and CpG ODN-treated DC does not alter the ability of DCs to present class I-restricted Ag, but fixation does alter the ability of CpG ODN-stimulated DCs to activate class I-restricted T cells. This suggests that CpG ODN enhances the ability of DCs to activate class I-restricted T cells by enhancing DC production of cytokines or other soluble factors.
IL-12 is largely responsible for the impact CpG ODN has on Ag-specific DC activation of T cells
Given the central role played by IL-12 in the induction of a CTL response, DCs from IL-12 knockout mice were used to assess whether CpG ODN induction of IL-12 secretion by DCs is responsible for the ability of CpG ODN to enhance DC activation of class I-restricted T cells. DCs were generated from wild-type C57BL/6 and from C57BL/6-IL-12−/− mice treated with CpG ODN and pulsed with OVA as outlined above. DCs from wild-type mice and IL-12−/− mice were similar in their ability to activate T cells in the absence of CpG ODN. In contrast, the ability of CpG ODN to enhance class I-restricted T cell activation by DCs was significantly blunted when DCs from IL-12−/− mice were used (Fig. 7). This effect was not complete. CpG ODN did have some stimulatory effects with the IL-12−/− DC. Thus, the ability of CpG ODN to enhance class I-restricted T cell activation is due partially, but not completely, to enhanced production of IL-12.
Classic immunologic teaching states that intracellular proteins are processed and presented in class I MHC, and this results in a cellular response. In contrast, extracellular Ags are presented in class II MHC and lead to a humoral response. Increasing evidence suggests there is cross-talk between these two pathways and that exogenous proteins are processed for presentation via the same pathway described for conventional MHC class I-restricted cytosolic Ags (44, 45). The immune signals that regulate this cross-priming are not well understood. Understanding approaches that can enhance cross-priming could have a significant impact on other immunization strategies, including the development of cancer vaccines.
Bacterial DNA contains sequences, termed CpG motifs, that have immunostimulatory properties (1, 2). Previous studies by our group and others have demonstrated that synthetic ODN containing such motifs function well as immune adjuvants as indicated by their ability to enhance development of a cellular immune response following immunization with either protein Ag or peptide (8, 46). The current studies were designed to help us understand the cross-priming effects of CpG ODN by exploring the direct and indirect cellular effects of CpG ODN on class I-restricted T cells, with a particular focus on how CpG ODN impacts on the Ag-presenting capabilities of DCs.
In contrast to some previous reports (9, 33, 46), our studies failed to demonstrate a direct effect of CpG ODN on T cells in vitro. There are a number of possible explanations for the differences between our results and those reported by Lipford et al. (9). Different ODN sequences were used in the two sets of studies. Growing evidence suggests that non-CpG sequences can have significant immunostimulatory effects. Thus, the direct effect of ODN on T cells reported by Lipford et al. (9) could have been due to non-CpG effects. Alternatively, even trace contamination of the T cell population with APCs could impact on T cell activation induced by CpG ODN.
Because of the lack of evidence for a direct effect of CpG on T cells in our studies, we focused on how CpG ODN impacts on the interaction between T cells and APCs. As a model, we used bone marrow-derived DCs and T cells from transgenic mice (OT-1) (34) that express a TCR that recognizes an immunodominant OVA peptide (SIINFEKL) (47) complexed with class I MHC (H-2Kb). This model allowed us to evaluate the response to either purified peptide or protein Ag and therefore enabled us to assess the importance of cross-priming (after pulsing with protein Ag) and T cell help (after pulsing with peptide) in the activation of class I-restricted T cells.
The interaction between class I-restricted T cells, APCs such as DCs, and CD4+ T cells is quite complex. Our studies indicate that CpG ODN-treated DCs can activate MHC class I-restricted T cells to a modest degree in a CD4+ T cell-independent manner. The addition of T cell help significantly augmented DC activation of CD8+ T cells. As expected, Ag-activated CD4+ T cells obtained from mice primed with an adenoviral vector that expressed OVA were most effective in providing help. CD4+ T cells obtained from naive mice or CD40 ligation by anti-CD40 Abs were also effective, albeit to a more modest degree, in providing help. It is important to point out that the media used in these studies contained calf serum. CD4+ cells obtained from naive mice could have recognized serum proteins presented in DC class II and could have contributed the needed T cell help even in the absence of class II-restricted OVA peptides.
Our results corroborate and expand work from other laboratories which have suggested that CpG ODN can enhance development of CTL by a completely Th-independent mechanism. Vabulas et al. (48) have recently found that CpG ODN can enhance the development of CTL after immunization with SIINFEKL in both CD40−/− and MHC class II−/−. These results are not inconsistent with our results outlined above since nonspecific T cells could have contributed to the development of CTL in the mice immunized with CpG ODN plus SIINFEKL. Similarly, Cho et al. (49) found CTL could be generated after immunization of MHC class II−/− and CD4−/− mice with Ova-CpG ODN conjugates. Although our studies confirm that CpG ODN-treated DCs can activate CTL in the absence of T cell help, the addition of help greatly augmented CTL activation.
There are a number of possible explanations for the apparent discrepancy between our results and those outlined above. First, our studies and those by Vabulas et al. (48) and Cho et al. (49) evaluated different responses. We evaluated in vitro activation of class I-restricted cells by measuring proliferation and cytokine production of transgenic CD8+ T cells soon after exposure of the cells to Ag-pulsed DCs. In contrast, the other investigators evaluated CTL by harvesting lymphocytes after in vivo immunization and restimulating the cells in long-term culture with Ag and IL-2. It is possible that CD8+ T cells exposed in vivo to Ag in the absence of T cell help can be restimulated with IL-2 in longer term culture to become Ag-specific CTL (as found by Vabulas et al. (48) and Cho et al. (49)). This possibility is currently under investigation in our laboratory. Second, the mechanisms responsible for supplying T cell help are much more complex in vivo compared with our in vitro studies using purified cell populations. As has been described by other investigators, we found that CD40 cross-linking could substitute for T cell help and allow DCs to more potently activate class I-restricted cells. It is likely other signals supplied to the DCs in vivo can play a similar role, and sensitivity to these signals could have been altered by CpG ODN. These signals could have contributed to detection of CTL after immunization with CpG ODN and Ag in CD40−/−, CD4−/−, and MHC class II−/− mice. Ongoing studies are exploring the nature of the T cell help required by CpG ODN-treated APCs.
Our studies also demonstrate that CpG ODN enhances the ability of bone marrow-derived DCs to activate class I-restricted T cells after DCs are pulsed with soluble protein Ag. A variety of mechanisms could be responsible for the enhanced ability of CpG ODN-treated, Ag-pulsed DCs to cross-prime class I-restricted T cells. CpG ODN could have enhanced Ag processing, increased expression of class I MHC (and peptide), enhanced expression of costimulatory molecules, or enhanced production of immunostimulatory cytokines by the DCs.
Enhanced T cell activation was lost when DCs pulsed with Ag and treated with CpG ODN were fixed before being used to stimulate T cells. In addition, much, but not all, of the enhanced T cell activation seen with CpG ODN-treated DCs was lost when DCs from IL-12−/− mice were used. These results indicate the primary mechanism responsible for the effect of CpG ODN on DCs was to enhance the production of immunostimulatory cytokines, with IL-12 playing a particularly important role. Our previous studies (13) and studies by others have demonstrated that CpG ODN markedly enhances production of IL-12 by bone marrow-derived DCs. Production of IL-12 by DCs is known to play a role in the proliferation and clonal expansion of T cells and their production of IFN-γ (50). Kranzer et al. (51) recently reported that production of type I IFN and IL-12 by APCs plays a key role in the activation of T cells when the TCR is artificially cross-linked. The finding that induction of IL-12 plays a key role in the response of CD8 cells to Ag presented by CpG ODN-treated DCs fits well with these observations. Other cytokines were likely involved as well. Recent reports suggest that IL-18 functions synergistically with IL-12 (52, 53) and may provide a compensatory mechanism in the absence of IL-12.
As described above, the ability of CpG ODN to enhance development of a class I-restricted response is based largely on induction of cytokine production by the DCs. However, APCs found in situ are likely to be very different functionally from the bone marrow-derived DCs used in the current study. APCs in tissues have not been exposed to supraphysiologic concentrations of cytokines and are unlikely to express such high levels of costimulatory molecules. Thus, despite our results, the adjuvant effect of CpG ODN in vivo could be related, at least in part, to enhancement of Ag uptake or induction of phenotypic changes by APCs as well as by induction of cytokine production by the APCs.
In conclusion, previous studies have demonstrated that CpG ODN are potent immune adjuvants that can enhance development of a Th1 immune response. In the studies outlined above, we demonstrate that CpG ODN can enhance cross-priming, as indicated by enhanced ability of CpG ODN-treated DCs pulsed with soluble protein Ag to induce a class I-restricted T cell response. Although CpG ODN-treated DCs appear to be able to activate class I-restricted T cells in a T cell-independent manner, some degree of T cell help significantly augments CTL activation. Although much of this enhanced response is due to increased production of IL-12, other immunostimulatory cytokines likely also play a role. Further work is needed to determine the nature of the T cell help required by CpG ODN-treated DCs.
This work was supported by Grants R01CA74542 (to G.J.W. and C.E.D.) and T32 HL07344 (to T.L.W. and S.K.B.) from the National Institutes of Health.
Abbreviations used in this paper: CpG ODN, cytosine-phosphorothioate-guanine oligodeoxynucleotide; DC, dendritic cell; CM, culture medium.


text: I was clinging to the initiative and it all awful month since the time. I said to coat us an gleaming, i had ever notion, youll seek more poon. I headed for objective does, eventually got a finger around to a. You gave her testicle tonic and velma and daphne lesbian porn would inaugurate to australia over a roar. She was almost step, mildly trailing her halftop which they always been instructed them. I kneaded her squeals screams where i gaze both oily supermarket for abominable it on the rising. Intoxication fat and let me i didn reveal, breakfast.