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SiriusXM Radio Reopens Studio 54 For 'One More Night'
September 13, 2011 at 3:51 PM (PT)
SIRIUSXM will bring together members of the original team that worked behind the scenes at STUDIO 54 during its 1977-1981 disco heyday to re-create what will be billed as "One More Night" inside the famed STUDIO 54 at 254 W. 54th Street in NEW YORK CITY. Studio 54 will reopen for the first time in more than 25 years, for one night only on TUESDAY, OCTOBER 18th.
Among the collaborators who'll recreate STUDIO 54's nightly parties for one more night are entertainment producer KARIN BACON, club architect SCOTT BROMLEY, doorman MARC BENECKE, former head of security CHUCK GARELICk and barman SCOTT TAYLOR, among others.
Original STUDIO 54 DJs NICKY SIANO and LEROY WASHINGTON will play the iconic era's dance classics. Celebrity guests, -- some from STEVE RUBELL's master call list -- and lucky SIRIUSXM listeners will be in attendance to flashback or dance for the first time in the internationally renowned club.
"There has been a great response to the launch of STUDIO 54 Radio and we want to continue that excitement with this special one-time event," SIRIUSXM Pres./Chief Content Officer SCOTT GREENSTEIN said. "We are working with many of the members of the original team that IAN SCHRAGER and STEVE RUBELL so brilliantly assembled to create one more night."
SIRIUSXM launched STUDIO 54 Radio, a 24/7 commercial-free channel devoted to the best classic dance and disco as a tribute to the music often played in the legendary club, on AUGUST 15th, 2011. STUDIO 54 Radio, channel 15, will broadcast live from the "One More Night" event that night.


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Puerto Rico Compania de Turismo
Tourism Company Puerto Rico
Download the vector logo of the Puerto Rico Compania de Turismo brand designed by in Encapsulated PostScript (EPS) format. The current status of the logo is active, which means the logo is currently in use.
- Designer:
- unkown
- Contributor:
- unknown
- Vector format:
- eps
- Status:
- Active Report as obsolete
- Vector Quality:
- No ratings
- Updated on:
- Mon, 01/26/2004 - 05:01

subset: commoncrawl


text:
Top 10 K-dramas to binge watch on Netflix in September
WRITTEN BY Aditi Shrivastava
Little women
On September 3, Netflix and tvN premiered Little Women, a K-drama based on Louisa May Alcott's book of the same name.
Once upon a small town
The novel of the same name by Park Ha-web Min served as the basis for the movie Once Upon A Small Town. Netflix will start streaming it from September 5.
Narco Saints
An upcoming criminal thriller on Netflix centers on businessman Kang In-su, who unintentionally ends up at the epicenter of a narcotics ring run by Koreans.
Extraordinary Attorney woo
An autistic lawyer joins a big law firm where she encounters a lot of challenges professionally and in love life. Park Eun Bin and Kang Tae oh star in the K-drama
Young lady and gentleman
Young Lady and Gentleman, a South Korean drama written and produced by Shin Chang-seok, stars Ji Hyun-woo, Lee Se-hee, and Cha Hwa-yeon.
Cafe Minamdang
A criminal profiler turns into a sahman after being falsely accused for his best friend's murder, later with his team he uncovers the truth.
My Liberation notes
Exhausted by the routine of adult life, three siblings look for happiness and escape from their routine lives.
Homemade love story
Over time, a number of individuals with distinctive personalities who reside at the Samgwang Villa grow to know one another.
The uncanny counter
Demon hunters pretend to be noodle restaurant employees in an effort to capture wicked spirits seeking eternal life.
Military prosecutor Doberman
Military Prosecutor Doberman, a 2022 Korean action, crime, and mystery drama directed by Jin Chang Gyu, is a gripping tale of good vs evil.
Netflix India top 10 trending web series of the week
Read More


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A very famous and locally recommended by many, Wei Ji Claypot Chicken Rice HERE, prepare you best Setapak claypot chicken rice. Notably its chicken meat is big portion and so much worth for a try ☺☺☺.
With only RM30.90, you will get 1 big size (for 2-persons) claypot chicken rice, added with sausage, and salted fish, and 3 drinks.
Address: 74, Jalan Mawar, Taman P Ramlee, 53000 Kuala Lumpur.
Contact: 016 252 7368


text:
Chelsea, SW3 is synonymous with exclusivity. Not because the community isn't welcoming (quite the contrary), but property prices make it prohibitively expensive for most people to live here.
However, to those who can afford the "barrier to entry", Chelsea offers ample opportunity for growth and leisure. Whether making contact with some of the most influential people in London or simply shopping in one of the many luxury stores, there's never a dull moment.
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A Fortune 500 Retailer, with a large distribution facility in Central Indiana, has partnered with NCW to assist with its ongoing staffing efforts. In this arrangement, NCW has been successful with meeting its ongoing needs by staffing Warehouse Material Handlers and Warehouse Associates for the 500,000 square foot distribution facility.
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Run your business on the go in the palm of your hand


text: Yes, I tried this second thing, but it doesn't work either. I created a new instance called test1.example.net. I created a volume snapshot (test1.example.net_ss1) I created a volume from that snapshot (vol_from_ss) I created a volume from the volume that was from the snapshot (vol_from_vol_from_ss) I deleted the vol_from_ss (this works) I deleted the snapshot (this does not work) If I then delete the vol_from_vol_from_ss volume, I can then delete the snapshot. P.S. My openstack cli says the option is --source, not --volume. Here is my command output. $ openstack volume list | grep test1 | cc698f20-c56f-47c7-b767-3c554c8cb517 | test1.example.net | in-use | 20 | Attached to test1.example.net on /dev/sda | $ openstack volume snapshot list | grep test1 $ openstack volume snapshot create --volume test1.example.net test1.example.net_ss1 Invalid volume: Volume cc698f20-c56f-47c7-b767-3c554c8cb517 status must be available, but current status is: in-use. (HTTP 400) (Request-ID: req-1ab60e86-3809-4a3b-9ac3-98e7d09ae7e9) $ openstack volume snapshot create --force --volume test1.example.net test1.example.net_ss1 $ openstack volume create --snapshot test1.example.net_ss1 vol_from_ss $ openstack volume create --volume vol_from_ss vol_from_vol_from_ss usage: openstack volume create [-h] [-f {json,shell,table,value,yaml}] [-c COLUMN] [--max-width <integer>] [--fit-width] [--print-empty] [--noindent] [--prefix PREFIX] [--size <size>] [--type <volume-type>] [--image <image> | --snapshot <snapshot> | --source <volume>] [--description <description>] [--availability-zone <availability-zone>] [--consistency-group consistency-group>] [--property <key=value>] [--hint <key=value>] [--multi-attach] [--bootable | --non-bootable] [--read-only | --read-write] <name> openstack volume create: error: unrecognized arguments: --volume vol_from_vol_from_ss $ openstack volume create --source vol_from_ss vol_from_vol_from_ss $ openstack volume delete vol_from_ss $ openstack volume snapshot delete test1.example.net_ss1 $ openstack volume snapshot list | grep test1 | 386186e9-6121-4222-b487-3dc026acf70d | test1.example.net_ss1 | None | available | 20 | $ openstack volume list | grep 'vol_\|test1' | ccbfdabe-d2ff-4bbd-a0ef-3b08935fe29a | vol_from_vol_from_ss | available | 20 | | | cc698f20-c56f-47c7-b767-3c554c8cb517 | test1.example.net | in-use | 20 | Attached to test1.example.net on /dev/sda | ---------------------------------------------------------------------------- ---- $ openstack volume delete vol_from_vol_from_ss $ openstack volume snapshot delete test1.example.net_ss1 $ openstack volume snapshot list | grep 'test1' $ openstack volume list | grep 'vol_\|test1' | cc698f20-c56f-47c7-b767-3c554c8cb517 | test1.example.net | in-use | 20 | Attached to test1.example.net on /dev/sda | --John -----Original Message----- From: Sean McGinnis <sean.mcginnis at gmx.com> Sent: Thursday, December 5, 2019 12:58 PM To: Ratliff, John <jdratlif at iu.edu> Cc: openstack-discuss at lists.openstack.org Subject: [External] Re: openstack volume snapshots and deleting This message was sent from a non-IU address. Please exercise caution when clicking links or opening attachments from external sources. ------- On Thu, Dec 05, 2019 at 03:24:35PM +0000, Ratliff, John wrote: > If I create a volume, snapshot it, and create a new volume from that > snapshot, I cannot seem to delete the snapshot until the volume is deleted. > Is this intentional, or am I doing something wrong? Is the new volume > based on the snapshot, and that's why I can't delete it? We're using > CEPH as a storage backend if that makes a difference. > That is the expected behavior as the snapshot is in-use by the volume created from it. So it can't be deleted until there isn't anything using it. Cloning would be the only way to get around this. There's not a quick and easy call to do this from a snapshot base, so unfortunately to get around that and get the final effect that it appears you are looking for, you would need to create a snapshot, create a volume from the snapshot, then *clone* yet another volume from that new one. Then delete the temporary volume and snapshot once the clone is done. As long as the backend driver is honoring the clone semantics, that should result in a fully independent volume that has its inital data set from the snapshot. So the process would look something like: openstack volume snapshot create --volume base-volume base-ss openstack volume create --snapshot base-ss temp-volume openstack volume create --volume temp-volume new-volume openstack volume delete temp-volume openstack volume snapshot delete base-ss Otherwise, it should be fairly safe to just keep the snapshot around. A lot of storage devices handle snapshots as internal pointers, so the only additional space consumption comes from anything new you end up writing to the new volume. Sean -------------- next part -------------- A non-text attachment was scrubbed... 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All of my ceramics are made from quality, high-fire porcelain clay. I use a Japanese style porcelain that contains no ball clay. This produces a very durable and beautiful finished piece. All of my pieces are thrown, glazed, and fired by me in my studio. Each piece is one of a kind. Even though I may use the same glaze on several pieces each one will come out different.
A little more about my process
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Hymnary.org will be unavailable January 30th, 6:00 to 9:00 PM EST for system maintenance. Thank you for your patience. Hide this message
"Crown Him Lord of all!" This joyful marriage of "All Hail the Power of Jesus' Name" with Twila Paris's contemporary praise song "He Is Exalted" sparkles with creative energy. Paris's song begins in a joyful 6/8 meter that continues throughout, transforming the hymn's entrance into a dance-like celebration. Optional brass and rhythm add the crowning touch to this refreshing blend of old and new.
Voicing: SATB,Pno


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He made the remarks on Tuesday, noting that the Taliban must show flexibility in the peace talks.
Afghan President Ashraf Ghani along with Mohammad Hanif Atmar is currently in Qatar to resolve a number of problems in Afghan peace talks.
Atmar maintained that one of the aims of this visit is to use the role of Qatar in the Afghan peace process and further expressed hope that the negotiations could lead to a ceasefire.
"Apart from Qatar, the US, Pakistan, Iran, Russia, and China have an important role to play in the talks, and the Afghan government is negotiating with all of these countries to achieve the desired result," he added.
Three weeks after the beginning of the inter-Afghan talks between the negotiating team of the Islamic Republic of Afghanistan and the Taliban, direct talks between the two sides have not yet taken place due to differences in the negotiating process.


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Zkittlez Regular Indica Marijuana Seeds
Enjoy a unique experience when you purchase ZKITTLEZ Regular marijuana weed seeds, a mostly indica strain, from Blimburn Seeds Bank.Our seeds are fresh and ready to germinate. Follow our Germination guide for best results!
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Anzellini, S ORCID: https://orcid.org/0000-0003-0091-3902, Errandonea, D, Burakovsky, L, Proctor, JE ORCID: https://orcid.org/0000-0003-3639-8295, Turnbull, R and Beavers, C 2022, 'Characterization of the high-pressure and high-temperature phase diagram and equation of state of chromium.' , Scientific reports, 12 (1) , p. 6727.
- Published Version
Available under License Creative Commons Attribution 4.0.
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The high-pressure and high-temperature phase diagram of chromium has been investigated both experimentally (in situ), using a laser-heated diamond-anvil cell technique coupled with synchrotron powder X-ray diffraction, and theoretically, using ab initio density-functional theory simulations. In the pressure-temperature range covered experimentally (up to 90 GPa and 4500 K, respectively) only the solid body-centred-cubic and liquid phases of chromium have been observed. Experiments and computer calculations give melting curves in agreement with each other that can both be described by the Simon-Glatzel equation [Formula: see text]. In addition, a quasi-hydrostatic equation of state at ambient temperature has been experimentally characterized up to 131 GPa and compared with the present simulations. Both methods give very similar third-order Birch-Murnaghan equations of state with bulk moduli of 182-185 GPa and respective pressure derivatives of 4.74-5.15. According to the present calculations, the obtained melting curve and equation of state are valid up to at least 815 GPa, at which pressure the melting temperature is 9310 K. Finally, from the obtained results, it was possible to determine a thermal equation of state of chromium valid up to 65 GPa and 2100 K.
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|Schools > School of Computing, Science and Engineering
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|Scientific reports
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|Generalitat Valenciana, Juan de la Cierva Formacion, Ministerio de Ciencia e Innovación, European Regional Development Fund
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text:
Examines how teacher preparation is contributing to deeper learning and to the development of 21st century skills
Offers a comparative analysis of six national-level professional development programs for teachers
Provides rich and nuanced descriptions and analyses of how these programs are designed and implemented in practice
Examines what large, ambitious national teacher development programs have in common, and discusses how they can enrich established ideas about what works in teacher education
Part of the book series: SpringerBriefs in Education (BRIEFSEDUCAT)
Table of contents (7 chapters)
Front Matter
About this book
This open access book presents a comparative study on how large-scale professional development programs for teachers are designed and implemented. Around the world, governments and educators are recognizing the need to educate students in a broad range of higher order cognitive skills and socio-emotional competencies, and providing effective opportunities for teachers to develop the expertise needed to teach these skills is a crucial aspect of effective implementation of curricula which include those goals.
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The ambitious education reforms studied here explicitly focus on building teachers' capacity to teach on a broader set of goals. Through a discerning analysis of program documents, evaluations, and interviews with senior leaders and participants in the programs, the book identifies the various theories of action used in these programs, examines how they were implemented, and discusses what they achieved. As such, it offers an indispensable resource for education leaders interested in designing and implementing professional development programs for teachers that are aligned with ambitious instructional goals.
- open access
- large scale professional development programs
- Teacher education and deeper learning
- Teacher education and twenty first century skills
- supporting teacher knowledge development
- supporting teacher pedagogical development
- designing teacher professional development
- implementing teacher professional development
- National education reforms and teacher professional development
- Comparative teacher education
- Mastery Math reform in England
- District based teacher professional development
- Teacher professional development in Colombia
- Teacher Professional development Mexico
- Teacher professional development in India
- Teacher professional development in Cambodia
- Teacher professional development in USA
"This book is a valuable resource to system leaders or policy makers keen to make change in their education or school system." (Pak Tee Ng, Educational Research for Policy and Practice, Vol. 19, 2020)
Editors and Affiliations
Graduate School of Education, Harvard University, Cambridge, USA
Fernando M. Reimers
About the editor
Bibliographic Information
Book Title: Empowering Teachers to Build a Better World
Book Subtitle: How Six Nations Support Teachers for 21st Century Education
Editors: Fernando M. Reimers
Series Title: SpringerBriefs in Education
DOI: https://doi.org/10.1007/978-981-15-2137-9
Publisher: Springer Singapore
eBook Packages: Education, Education (R0)
Copyright Information: The Editor(s) (if applicable) and The Author(s) 2020
License: CC BY
Softcover ISBN: 978-981-15-2136-2Published: 24 March 2020
eBook ISBN: 978-981-15-2137-9Published: 23 March 2020
Series ISSN: 2211-1921
Series E-ISSN: 2211-193X
Edition Number: 1
Number of Pages: VIII, 135
Number of Illustrations: 1 b/w illustrations
Topics: Teaching and Teacher Education, Educational Policy and Politics, Curriculum Studies, Education Policy, Sociology of Education


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Île-de-France1 - 1 of 1 listings
wanted: Paris Oktober/NovemberCheck with sellerApartments Paris (Île-de-France) August 16, 2021Hi! I'm looking for an apartment for Oktober and November in Paris, preferably in the city. Part-time (50/50 a month) would also work, in that case I'd spend the other half at PAF.
- 1


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Find answers to common questions about GDC
See answers to some of the most frequently asked questions regarding GDC.
If you still have a question that you can't find the answer to, please contact us.
GDC 2023 Event Details
When is the Game Developers Conference 2023?
GDC 2023 takes place from March 20 through March 24, 2023.
Where will the Game Developers Conference 2023 be located?
GDC 2023 will be held at the Moscone Center in San Francisco.
When and where can I pick up my GDC Badge?
Registration location and hours for GDC 2023 will be posted closer to the event.
How much does it cost to attend GDC?
GDC offers a variety of passes, with prices depending on the level of access. For details, visit the Passes & Prices page.
Do I need to be vaccinated to attend GDC in person?
Yes, proof of COVID-19 vaccination or a negative COVID-19 test result will be required to attend GDC 2023. Masks will not be required but are strongly encouraged. The event will also feature fixed hand sanitization units throughout the facility, contactless registration, and enhanced cleaning. You can see all our new initiatives designed to keep you safe on the Health & Safety page.
Where can I find my invoice, receipt, and/or confirmation after registering?
After you register and pay, you will receive your receipt at the email address you used to register from [email protected]. If you registered as part of a group, your group organizer may have your receipt and confirmation. Email GDC Registration for assistance.
How do I register a group?
Groups of 10 or more attendees registering at the same time can save 10% on the current registration price for the All Access Pass, Core, or Summits Pass types. Additional discounts, such as Alumni, exhibitor, or other special discounts are not combinable with the group discount. Additional registrations by the same group organizer at a later date can receive a 10% discount on the current registration price at the time of submission.
If you are interested in purchasing 10 or more GDC passes, visit Group Registration.
When/where do I pick up my GDC badge?
Please pick up your badge in person, on-site at GDC. A valid photo ID, the QR code printed on your confirmation email and proof of vaccination or negative COVID-19 test result will be required to pick up your badge. Collecting multiple badges by a single person is not allowed. Badges will not be mailed in advance of the event.
PLEASE TREAT YOUR BADGE AS CASH: Your badge is required for entry at GDC. A non-refundable replacement fee equal to the original purchase amount will apply to all lost, duplicate, misplaced, stolen, or forgotten badge/badge holder requests. If your badge was complimentary, the fee will be the current on-site rate.
What is the cancellation policy?
To cancel, change, or substitute your pass, please email GDC Registration.
Requests for registration refunds or substitutions must be received before February 22, 2023. Refunds, transfers or substitutions are not available after this date, are not available for no-shows, and will not be offered due to lack of or inability to provide proof of vaccination at the event. Paid registrants who cancel or downgrade and do not substitute another person will receive a full refund less a 6% processing fee if notification is submitted in writing to GDC Registration on or before February 22, 2023.
If you have any questions, please contact GDC Registration.
Do international attendees need a visa to attend GDC?
Maybe. Please view the United States visa requirements. You can request a letter of invitation when purchasing a conference pass. Your letter of invitation will be attached to your confirmation email.
How do I obtain a Letter of Invitation?
If you require a Letter of Invitation to obtain a visa, you can request it during online registration. Letters of Invitation will only be issued in the name of the registrant and only after the registration is paid in full.
Can I contact a live person for registration help and information?
Please email GDC Registration or call (415) 947-6926 or (866) 535-8997 for assistance and information.
GDC Masterclass
What is the GDC Masterclass program?
The GDC Masterclass program is comprised of day-long or multi-day, small-group workshops that deliver in-depth, hands-on training around some of the most important challenges facing game developers today.
When will the next round of GDC Masterclass courses be offered?
Registration is now open for GDC Masterclass courses taking place on November 3-4 & December 8-9, 2022. All courses will be offered in November with a repeat offering in December, please read each description carefully and choose the course dates that work best for you when registering.
How will I access the GDC Masterclass program?
Virtual GDC Masterclass courses will be hosted on Zoom. After registering, you will be sent instructions on how to use and access the platform via email in the weeks leading up to your course.
Diversity & Inclusion
Are there complimentary or discounted passes?
GDC provides limited discounts to sponsors, media partners, community organizations, low income groups, and association partners. More information on these programs can be found on our Diversity & Inclusion page.
How do I apply to become a Conference Associate at GDC?
GDC Conference Associates receive a complimentary All Access pass in exchange for 25 hours during GDC week answering attendee questions, monitoring doors, checking badges, and other responsibilities. Learn more about the GDC Conference Associate program on our Diversity & Inclusion page and apply to participate.
2023 CA call for applications are open through December 22, 2022. Click here to submit an application if you are interested.
Does GDC have a code of conduct?
Yes. GDC is committed to providing a friendly, safe, and welcoming environment for all regardless of gender identity, sexual orientation, disability, ethnicity, or religion. The GDC Code of Conduct outlines expectations for participant behavior and consequences for unacceptable behavior.
How can I request assistance in accordance with the Americans with Disabilities Act?
If you require special assistance, auxiliary aids, interpreters or other reasonable accommodations while at the conference and/or expo, please contact Michelle Montoya at least four weeks prior to the event date.
Does GDC provide childcare services?
Yes. While no one under the age of 18 (including infants in strollers) is allowed to attend the conference or expo, GDC provides on-site childcare services. Please visit the KiddieCorp website for more information and to register for childcare services.
Does GDC provide a private room for nursing mothers?
Yes. Nursing mothers may use the private Mothers Lounge located on-site.
Other Questions
When/where will conference sessions dates and times be available?
Dates and times for 2023 conference sessions will be added to the GDC website about 4 weeks before the event.
What speaking opportunities are available at GDC?
Core Concepts Call for Submissions closed August 16, 2022.
GDC Summits and Game Career Seminar Call for Submissions closed October 7, 2022.
GDC Masterclass submissions are accepted year round, please contact Sam Warnke to be considered.
To stay up to date on information about speaker proposals and submission deadlines, subscribe to our Call for Submissions mailing list here!
Can I watch GDC sessions after the event?
Yes, GDC records most sessions and makes them availible to view online throughout the year on GDC Vault.
Attendees with an All Access pass receive access to GDC Vault through March 2024. Alternatively, you can purchase an individual or studio membership to access GDC Vault.
Is there Internet access available at GDC?
Yes. Wi-Fi is available in the lobbies and session halls of the Moscone Center during the event, but not in the GDC Expo. There is no technical support for Wi-Fi at GDC.
What are the filming and recording guidelines?
Legitimate news media with approved credentials and media badges whose only intention is to gather film clips for a newscast can receive special clearance to film onsite at the Moscone Convention Center, with restrictions.
Please note that there are limitations (even for media badge holders) for filming or recording at the Moscone Center, and filming or recording of GDC content/sessions is strictly prohibited.
Documentary film crews are not considered legitimate news media and therefore union regulations require hiring a Local 16 IATSE representative, in addition to gaining filming approvals from the GDC organizers via the GDC Press Team.
GDC reserves the right to approve all filming and recording within the Moscone Center during the event. Learn more about filming guidelines, including how to hire a labor technician/union representative.


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We start with design; then move on to discuss the latest hot topics. It's a podcast where we like to share our POV & listen to others. This is a platform that is designed to inform & educate.
Design Perspectives Gail M Davis
- Arts
We start with design; then move on to discuss the latest hot topics. It's a podcast where we like to share our POV & listen to others. This is a platform that is designed to inform & educate.
Here are a few questions that I had for Amy:
What is my "why?"
I am a creative person and a business owner and I understand the necessity and the difficulties of communicating your own story well. Given my background in editorial, I felt I could demystify and simplify what it means to be "press ready" from that perspective, which is just one perspective but an important one when trying to get your message out there!
Who is my clientele?
Mainly interior designers. Increasingly small manufacturers and brands in the interiors industry. The communications component that is now so closely bound to business continues to express itself in new ways within the design community, whether with showrooms, design centers, or large design markets.
How to be "pitch perfect"?
First, understand that writers and editors look for the special in your work, a point of view. You can learn what it means to communicate that in my book, Recipe for Press: Designer Edition.
But in a nutshell, pitching requires clarity, reliability, consistency and reciprocity. It is a process that takes time and valuing the process of creating relationships within the design community.
Elevator pitch in three seconds.
You be you, with conviction! I treasure people's story. I always have.
This is the kitchen renovation episode! My kitchen renovation to be exact! I have been toiling away for the past 4 months working on my kitchen. I have learned A LOT and will share my journey. This kitchen was designed with the grown & sexy in mind! I want it to feel like you are experiencing your favorite restaurant whose dining area is sultry & moody. I wanted it to be a visceral tactile experience.
Thank you to House Beautiful and the following sponsors for being so AMAZING to work with:
Emtek - https://www.emtek.com/
Monogram - https://www.monogram.com/
The Tile Shop - https://www.tileshop.com/
Fabuwood - https://www.fabuwood.com/
Leeor Zorel - https://www.thezorelgroup.com/
Anne started her eponymous firm after relocating to Toronto 15 years ago after working for Daniel Romuladez on all Tory and Chris Burch's projects. She was House & Home's Designer of the Year in 2021 and has been featured in House Beautiful (her home was the cover story), The Wall St Journal, AD, Domino to name a few. She just released her first ever design and lifestyle book, MOOD, which is available on Amazon and in select stores.
More about MOOD
In Anne Hepfer's world, a home is a complex reflection of who you are, and the people, places and ideas that matter most. For this, her first, book, the acclaimed designer explores the power of a stylish and soul-nourishing refuge of one's own. Drawing inspiration from influences as wide-ranging as travel, music, food and drink, fashion, and nature, Hepfer shares her process and opens the doors to the masterful spaces she has crafted for both international clients and her own family. Filled with exuberant color and meditative reflections, MOOD is an ever-evolving journey through the seven key emotions a brilliantly designed home should evoke: happy, relaxed, energized, cozy, sexy, tranquil, and nostalgic.
Liz Lidgett's passion for art allowed her to see that competent art consulting for private companies and corporations was a need that wasn't being filled. With that in mind, she began her art consultation business, Adore Your Walls, in 2013. Since then, her business has grown and she recognized once again that online and national consulting – the type that exists for furniture and interior design – was completely absent for the world of art and has expanded her services, including a gallery space. With her developed expertise, Adore Your Walls has evolved into her new brand: Liz Lidgett Gallery + Design. Within the rebrand, Liz continues to consult, partner with artists and regularly contribute to design publications across the country.
Co-founded in 2007 by interior designers Beth Dotolo and Carolina V. Gentry, Pulp Design Studios is an iconic force to be reckoned with in the world of interiors, and the firm brings a sense of adventure to the business of design.
With a forward-thinking approach to commerce and design, Pulp has established a unique viewpoint that combines luxury design and a flair for the unexpected. With studios in Seattle, Dallas, and Los Angeles, the Pulp team works with residential and commercial clients across the country, helping them define a personal style and transforming interiors into spaces that are brag-worthy and innovative.
Episode 120- Gail M Davis
Why do you attend conferences? Are you going to truly learn? What are you expecting to glean? Do you want to learn how to get better? Or do you want someone to do the work for you? I share my perspective on what it is like to experience a conference as a speaker instead of being in the audience.
Customer Reviews
Episode 97
This episode really spoke to me. It solidified many thoughts I had been having lately, but not taking seriously. Thank you for this affirmation. ❤
Joyful and Real
Wha a joy you are, Gail! Thanks for keeping it real and sharing all of your wonderful knowledge and point of view.
Exquisite Podcast, Fantastic Host and Great Conversations!
I have met Gail as a client initially and we have developed a good friendship since! She is so knowledgeable about the industry and listening to her podcast has added so much value to our company and to me personally. She keeps us up to date with the latest trends, important figures in the industry and of course her 'Design Perspective' ;)
I was lucky enough to be interviewed by Gail in Aug 2021 and I immediately felt like I was talking to a close friend. We talked about different topics and as always she had an interesting perspective about the industry. She not only showed me that she is an experience interviewer but also made me feel very comfortable.
She has interviewed great designers, all with a great attitude and willing to spend a great and honest conversation.
Definitely I'll keep listening… Thank you Gail!


text: I read a lot on the forum about Grade 6, 7, etc. Someone will post "I just passed Grade 6!" I don't think we have this kind of standard grading system here in the States. Is this grading system specific to Britain? Or is it one of those things like the metric system where the rest of the world uses it and we're just being stubborn about adopting it?


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DETECTIVES have released CCTV footage of a man they want to speak to in connection with a robbery.
A 51-year-old man from Stanford le Hope was in Market Square, Basildon, at 1.20pm on Monday, February 1 when he was approached by a man who asked for a small amount of money for a bus fare.
When the victim took out his wallet, the suspect demanded money and grabbed the wallet from his hands before running off towards the bus station.
The victim was not injured but was left shaken by the incident.
Enquiries are ongoing and police are now in a position to release footage of a man they want to identify.
He is described as black, about 6ft tall, of medium build and wore a dark baseball cap, dark jacket and dark trousers with a white emblem on the left leg.
Anyone with information is asked to contact Dc Steve Dawkins at Basildon CID on 101.
Alternatively, they can contact Crimestoppers anonymously on 0800 555111 or www.crimestoppers-uk.org


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Vintage 80s USS Pennsylvania Submarine Tan Graphic T Shirt Unisex Small-Medium
Product Details
Killer 80s USS Pennsylvania submarine tan graphic t shirt. The USS Pennsylvania is among the largest subs ever made by the US Military, and are nuclear powered! Tagged a Medium, fits a Small to Medium. 50/50 blend, single stitch. Great vintage condition, no flaws to note. This tee might be from when this sub was launched back in '89, so rad!
Pit to pit = 18.5"
Back collar to bottom = 25.5"
Please check all measurements and photos. This is a vintage as/is listing. All sales final.
Care/Washing Instructions for Vintage T Shirts: Hand wash cold or machine wash cold on a Delicate setting. Hang dry.


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Vietnam: Fighting the Second Wave
In July 2020, a second wave of COVID-19 hit the city of Da Nang, Vietnam. A team from Hanoi's Bach Mai hospital who survived lockdown in the first wave came to support their colleagues and share their expertise on preventing in-house infections. But with far fewer resources, staff and equipment, it's a tough battle. To prepare for the worst-case scenario, they transform a local sports stadium into a field hospital. Follow the hard work of the medical team as they fight a multi-front battle.
Bach Mai hospital's veteran nursing director gives instructions to an ICU nurse. The hastily assembled nursing team is an assortment of workers from different regions and departments.
A sports stadium was hastily transformed into a field hospital to deal with a worst-case scenario.
A hospital worker takes a brief rest in a rare moment of downtime during a month-long battle.


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Data Engineer 0403
- Employer
- Location
- London (Central), London (Greater)
- Salary
- Up to £575 / day (Pro Rata)
- Closing date
- 30 Oct 2022
View more
- Contract
- Temp
- Hours
- Full time
- Listing type
- Job vacancy
- Industry
- Arts & heritage, Music
- Job function
- Data
- Job level
- Management
Our client, a global media company, is seeking a highly competent Data Engineer for a temporary contract.
Key responsibilities will include:
• Working on the expansion and management of the team's modelling infrastructure and optimising model pipelines.
• Developing and supporting the data and analytics team's machine learning tech stack.
• Enabling customers to access model results in a timely fashion.
• Leading on the improvement of existing models with thin team using state-of-the-art modelling techniques and tools.
• Substantial relevant experience in a similar role.
• Work experience using AWS (Sagemaker, Redshift, Glue.Lamda, Airflow).
• Comprehension of Machine Learning techniques and technologies, to include Regression, Tree based models, Neural Networks, and Deep Learning.
• Robust knowledge of Python and SQL.
• Experience contributing to the architecture and design of new and existing systems.
• Experience working with text data and NLP techniques is desirable.
• Comfortable working autonomously.
Please note that due to the volume of submissions, only successful candidates who meet the exact requirements will be contacted. If you have not heard back within 10 days of submission, please assume that your application has been unsuccessful on this occasion.
The Music Market/Arts and Media are an equal opportunities employer who supports diversity in the workplace.
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Pathogenic potential of IgG binding to water channel extracellular domain in neuromyelitis optica
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Background: Autoantibody specific for the aquaporin-4 astrocytic water channel is restricted to serum and CSF of patients with neuromyelitis optica (NMO) and related CNS inflammatory demyelinating disorders (relapsing optic neuritis and longitudinally extensive transverse myelitis). NMO-typical lesions are distinct from MS-typical lesions. Aquaporin-4 is lost selectively at vasculocentric sites of edema/inflammation coinciding with focal deposits of immunoglobulins (Ig) G, M, and terminal complement products, with and without myelin loss. Evidence for antigen-specific autoantibody pathogenicity is lacking.
Methods: We used confocal microscopy and flow cytometry to evaluate the selectivity and immunopathological consequences of Ig binding to surface epitopes of living target cells expressing aquaporin-4 fused at its cytoplasmic N-terminus with GFP. We tested serum, IgG-enriched and IgG-depleted serum fractions, and CSF from patients with NMO, neurologic control patients, and healthy subjects. We also analyzed aquaporin-4 immunoreactivity in myelinated adult mouse optic nerves and spinal cord, and plasma cell Ig isotypes in archived brain tissue from an NMO patient.
Results: Serum IgG from patients with NMO binds to the extracellular domain of aquaporin-4; it is predominantly IgG1, and it initiates two potentially competing outcomes, aquaporin-4 endocytosis/degradation and complement activation. Serum and CSF lack aquaporin-4-specific IgM, and plasma cells in CNS lesions of NMO contain only IgG. Paranodal astrocytic endfeet highly express aquaporin-4.
Conclusions: NMO patients' serum IgG has a selective pathologic effect on cell membranes expressing aquaporin-4. IgG targeting astrocytic processes around nodes of Ranvier could initiate demyelination.
- aquaporin-4;
- green fluorescent protein;
- human embryonic kidney cell line;
- multiple sclerosis;
- neuromyelitis optica.
Inflammatory demyelinating diseases of the central nervous system (CNS) are a major cause of nontraumatic neurologic disability in young adults, and they are recognized to be immune-mediated. The most common clinical diagnosis is multiple sclerosis (MS), which lacks a specific biomarker. We recently defined an autoantibody marker that is specific for neuromyelitis optica (NMO, also known as optic–spinal MS) and related disorders. NMO-IgG is the first autoantibody marker for any form of idiopathic CNS demyelinating disorder. It binds selectively to aquaporin-4 (AQP4), the most abundant water channel in the CNS.1–5 AQP4 is not a component of myelin or oligodendrocytes but, rather, an integral homotetrameric protein that is highly expressed in the polarized plasma membrane of astrocytic endfeet associated with CNS microvessels, ventricular ependyma, and interneuronal synaptic junctions. AQP4 regulates bidirectional water flux between blood and brain and between the brain and spinal fluid that accompanies K+ ion fluxes.6,7
Detection of NMO-IgG allows distinction of NMO from relapsing–remitting MS, with which it is often confused.1,3–5,8 NMO is recognized traditionally by a selective involvement of the spinal cord (typically longitudinally extensive lesions) and optic nerve,8 severe clinical morbidity, and destructive immunopathological lesions that affect both gray and white matter.9 Clinical and pathologic observations support a pathogenic role for an autoantibody of AQP4 specificity in NMO: 1) patients with acute NMO respond favorably to antibody-depleting therapies10,11; 2) brain MRI lesions surrounding the ventricles, which are typical of NMO,12 localize at sites of high AQP4 expression13; 3) CNS lesions in NMO exhibit loss of AQP4 immunoreactivity14,15; and 4) CNS lesions in NMO exhibit deposition of immunoglobulins (IgM and IgG) and products of complement activation in a vasculocentric pattern that coincides with AQP4's normal distribution.9,15
This report addresses three questions concerning the pathogenesis of NMO raised by the identification of AQP4 as a disease-specific antigen2: 1) What are the immunobiological outcomes of immunoglobulin interaction with AQP4 in target cell membranes? 2) What classes of AQP4-specific Igs have pathogenic potential? and 3) What accounts for loss of myelin (an oligodendrocyte product) if NMO is an autoimmune water channelopathy targeting astrocytes?
Cell lines and transgenic constructs.
Major binding sites of NMO-IgG (and affinity-purified, AQP4-specific rabbit IgG) in sections of normal adult human15 and mouse1,2 CNS tissues are where AQP4 is expressed in highest density, namely, in the dystroglycan complex of the highly polarized plasma membrane of astrocytic foot processes.7 AQP4 is readily visualized at those sites immunohistochemically because of its ultrastructural organization at those sites in tightly packed arrays.16 In preliminary Western blot studies (with protein-overloaded conditions), we observed that the rat oligodendroglial–astrocytic progenitor cell line (CG4),17 when differentiated in vitro to astrocytic phenotype (stellate and expressing prominent cytoplasmic GFAP intermediate filaments), expresses minimal AQP4 immunoreactivity, and that two independent human astrocytoma cell lines (HTB-14 and CRL-1718; obtained from the American Type Culture Collection) did not express detectable AQP4 by immunofluorescence or Western blot. Therefore, as the initial system to investigate in vitro the immunobiological consequences of IgG interacting with extracellular epitopes of AQP4, we chose to use our previously described, transfected human embryonic kidney cell line (HEK293) overexpressing full-length human AQP4 protein (fused at its N-terminus with green fluorescent protein [GFP]).2 The parental HEK293 cell line does not express AQP4 constitutively, but it does express dystroglycan complex partner proteins of AQP4, which ensure stable AQP4 insertion in the plasma membrane.2 Furthermore, the proximity of adjacent AQP4 homotetramers in the transfected cell's membrane mimics the close packing of epitopes in the highly polarized astrocytic foot process.16 (See Complement activation results section for discussion of other practical advantages of using AQP4-transfected HEK293 cells as substrate.)
Antibodies and sera.
Fluorochrome-conjugated goat IgGs were purchased from Molecular Probes (Alex-Fluor 546: human, mouse, or rabbit IgG specific; and Oregon Green: mouse IgG specific) or Invitrogen (Cy5; rabbit IgG specific). Conjugated monoclonal mouse IgGs (FITC: specific for human IgG subclasses; or Cy3: specific for glial fibrillary acidic protein and sodium channel) were from Sigma, and unconjugated IgGs were from BD Biosciences (Caspr1 and early endosome antigen-1 [EEA1]) and Dako, Denmark (human CD138). Goat IgGs monospecific for human IgG (TRITC conjugated) or human IgM (FITC conjugated) were from Southern Biotechnology. Rabbit IgG specific for AQP4 residues 249-323 was from Sigma. Rabbit IgG specific for human C9neo was a gift from Dr. Paul Morgan, Cardiff, UK. Deidentified patients' sera were obtained from the Neuroimmunology Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minn.
All final preparations of live cells, and sections of mouse tissue and autopsied human lymph node and brain tissues, were mounted in ProLong Gold DAPI antifade medium (Molecular Probes). Fluorescent images were captured from a Zeiss LSM510 confocal microscope. Immunoperoxidase-stained sections were counterstained with hematoxylin and were photographed from an Olympus DP-BSW microscope and DP70 digital camera system.
Live cells were exposed sequentially to 20% human serum or rabbit anti-AQP4 IgG diluted 1:1000, a secondary antibody (1:500), and chilled 95% ethanol/5% acetic acid (15 minutes). Cells fixed in 10% formalin were permeabilized, blocked, and incubated in primary and secondary antibody probes. Tissues dissected from perfused mice were postfixed overnight in 4% paraformaldehyde, held 24 hours in 30% sucrose, frozen in OCT medium, sectioned (8 μm), air-dried, and incubated sequentially in 10% normal goat serum containing 0.1% triton X-100 (30 minutes), primary antibody (18 hours), and secondary antibody (1 hour; room temperature). Autopsied human lymph node and brain tissues, fixed in 10% formalin and embedded in paraffin, were deparaffinized, sectioned (5 μm), washed in bidistilled water, and steam heated. Sections were incubated with 10% normal goat serum (1 hour) and then overnight at 4°C with primary antibody (anti-CD138 at 1:50; anti-IgG at 1:750; anti-IgM at 1:200).
Antigen modulation assay.
Complement was inactivated in the serum of patients and control subjects by holding 30 minutes at 56°C. Time-lapse photomicrographs of living cells were captured at 15-minute intervals for 24 hours. Media containing human serum were replaced at 16 hours with fresh media, with or without cycloheximide (0.25 μg/mL). Test sera were added to the culture media for either 16 hours at 37°C or for 1 hour at 4°C. Cells were washed, exposed for 30 minutes to Alexa-Fluor 546-labeled anti-human IgG (1:500), washed again, fixed in chilled 95% ethanol/5% acetic acid, rinsed in PBS, and stained with Hoescht (1 μg/mL).
Complement activation assays.
GFP-AQP4-HEK293 cells were incubated for 2 hours on ice with 20% heat-inactivated control or NMO patients' serum. Cells were washed with chilled DMEM media and exposed to 20% fresh (or heat inactivated) normal human serum (as the complement source) for 45 minutes at 37°C. After rinsing and fixing (10% formalin, 4 minutes), cells were incubated sequentially, at room temperature, in 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (1 minute), 10% normal goat serum and rabbit anti-human C9neo antibody (30 minutes), washed and incubated in labeled anti-rabbit IgG. To evaluate complement's effects on membrane integrity, test pools of human serum were added to the cells at 4°C (for 1 hour). After subsequent incubation for 90 minutes at 37°C (95% air/5% CO2) in fresh or heat-inactivated Low-Tox-H rabbit complement (Cedarlane Lab; 20% final concentration), the monolayer was examined and photographed by transmitted bright-field imaging. To quantitate complement-mediated membranolytic activity by flow cytometric analysis after sequential exposure to serum (30 minutes at room temperature) and complement (37°C for 30 minutes), the cells were lifted with trypsin, washed, suspended in chilled buffer (10 mM Hepes/NaOH, pH 7.4; 140 mM NaCl; 2.5 mM CaCl2) containing 0.5 μg of propidium iodide (BD Biosciences), and held in the dark for 15 minutes before analysis. Increase in membrane permeability was determined by comparing the percentage of propidium iodide–permeable GFP-positive cells with that of propidium iodide–permeable GFP-negative cells. In the absence of antigen-specific activation of complement, the increase in membrane permeability was less than 1.25-fold (125%).
IgG depletion and purification.
IgG was depleted from serum pools using protein G-agarose beads (2 hours at 4°C). IgG-depleted serum was used at a concentration of 20% in the complement assay. IgG eluted from washed protein G beads in 0.1 M acetic acid was neutralized, dialyzed against PBS, and concentrated using Centricon YM-3 filter devices (Millipore). IgG concentrations were determined by rate nephelometry.
Time-lapse photomicrographs.
GFP-AQP4-HEK293 cells were seeded onto optical-quality glass-bottom plates (MatTek) and exposed to 20% control or NMO patients' serum (see video E-1 on the Neurology® Web site at www.neurology.org). Time-lapse photomicrographs of the living cells were captured at 15-minute intervals for 6 hours, using a Zeiss LSM510 microscope with the stage held at 37°C in 95% air/5% CO2. Z series imaging was performed for each time point and converted into video.
NMO-IgG initiates endolysosomal degradation of AQP4.
A prerequisite for IgG to effect organ-specific pathogenicity is its capacity to bind to specific epitopes accessible on the surface of living target cells. When we exposed transfected target cells expressing surface AQP4 to a pool of NMO patients' sera (at 4°C to restrict membrane fluidity), IgG bound to the plasma membrane in a linear pattern that colocalized with GFP-AQP4 (figure 1A). Serum IgG from control patients did not bind detectably to the cell surface, and a control rabbit IgG specific for AQP4 cytoplasmic epitopes did not bind unless the plasma membrane was permeabilized. NMO patients' serum IgG did not bind to vector-transfected control HEK293 cells (data not shown). Thus, NMO patients' IgG binds specifically to the extracellular domain of AQP4.
To evaluate the influence of this IgG on plasma membrane AQP4, we recorded the distribution of GFP-AQP4 after exposure to patients' serum at 37°C. Control human serum had no effect on GFP-AQP4, but NMO patients' sera containing AQP4-specific IgG resulted in rapid disappearance of GFP-AQP4 from the plasma membrane (figure 1B and video E-1). The phenomenon's temperature dependence (figure 1B) is consistent with modulation of surface antigens through intermolecular cross-linking by bivalent IgG.18 Serial confocal images of living cells held at 37°C for 16 hours in serum lacking AQP4-IgG were indistinguishable from images of untreated cells with respect to GFP-AQP4 fluorescence intensity and localization (figure 2A). Cells exposed to NMO patients' serum underwent a striking redistribution within 5 minutes. The homotetrameric structure of AQP4, with multiple repeated epitopes in the extracellular domain, and the proximity of individual AQP4 homotetramers, provides an optimal target for rapid surface clearance from the plasma membrane by antigen-specific IgG. Plasma membrane fading was accompanied by green fluorescent vesicle accumulation in subplasmalemmal cytoplasm. After 20 minutes, GFP-AQP4 aggregated in ring-like structures. At 5 hours, when plasma membrane fluorescence was no longer detectable, these had coalesced into large, cytoplasmic aggregates. Removal of the patients' serum at 16 hours resulted in rapid reappearance of GFP-AQP4 in subplasmalemmal vesicles, presumably en route from the endoplasmic reticulum (figure 2B). By 4 hours, GFP-AQP4 was visible in the plasma membrane, and by 12 hours it had attained normal surface density. When cycloheximide was added at the time of withdrawing patients' serum, no GFP-AQP4 was seen on the plasma membrane during 12 hours of observation. This implies that the "reversal" of the autoantibody's effect was dependent on new protein synthesis.
We followed the fate of GFP-AQP4 cleared from the plasma membrane after exposure to NMO patients' serum by probing with an antibody specific for an early endosome antigen. In the absence of serum, early endosomal vesicles (red) did not colocalize with GFP-AQP4 (figure 2C). However, after 30 minutes of exposure to NMO patients' serum, early endosomal vesicles acquired green fluorescence (figure 2C). Early endosomal vesicles did not acquire green fluorescence when cells were exposed for 2 hours to control sera (data not shown). These results indicate that endocytosis, initiated by the cross-linking of AQP4 by IgG binding at the cell surface, targets AQP4 to the endolysosomal pathway for degradation.
NMO-IgG initiates complement activation.
Activation of the classic complement cascade is another potentially pathogenic outcome of autoantibody binding to AQP4. This mechanism would increase the permeability of microvascular endothelium, promote inflammatory cell infiltration, and possibly inflict focal damage to astrocytic endfeet in CNS regions enriched in AQP4. We first evaluated surface membranes of transfected cells by indirect immunofluorescence for deposition of the C9neo epitope created by the polymerization of multiple perforin-like C9 molecules in assembling the terminal membrane attack complex of complement.9,19,20 In the presence of NMO patients' sera and active human complement, C9neo was clearly visualized in the plasma membrane of HEK293 cells expressing AQP4 (figure 3A, arrows), but not in the absence of disease-specific serum or in the presence of inactive human complement, or in control cells transfected with GFP vector (data not shown). These data indicate that NMO patients' sera contain an AQP4-specific antibody capable of activating the complement cascade. Reagents specific for human IgG subclasses revealed that AQP4-specific serum IgG was, in all NMO-IgG–positive patients tested, exclusively IgG1, which is a major complement-activating IgG subclass in man (figure 3B).
Preliminary experiments indicated that membrane lysis conditions had to be rigorously controlled to evaluate functional evidence for antigen-specific complement activation by NMO-IgG on membranes of living cells expressing AQP4 in high density. The GFP-AQP4 transfected target cell line was a mixed population of GFP-positive (AQP4+) and GFP-negative (AQP4−) cells (figure 3C). In the presence of control human serum, phase microscopy revealed healthy AQP4+ and AQP4− cells in an adherent monolayer. When we preexposed the target cells to disease-specific patients' serum (on ice for 2 hours) before adding complement for 90 minutes at 37°C (conditions used for the C9neo visualization), all green cells disintegrated rapidly. We therefore used a standardized commercial preparation of rabbit complement, in limited concentration, and we preexposed the cells to patient or control serum at 4°C for just 1 hour before adding fresh or heat-inactivated complement. In the presence of NMO patients' serum, adherent cells that visibly expressed GFP were selectively lost. Provided that complement was active and present for more than 30 minutes, most AQP4+ cells were rounded and floating (figure 3C). Control human serum did not visibly affect either adherent population, regardless of complement activity. Thus, the cytolytic activity of NMO patients' serum depended on both complement activity and AQP4 expression in target cells.
Quantitation of target cell membrane lesioning by complement, using flow cytometric analysis (indicated by permeability to the red dye propidium iodide), necessitated further attenuation of complement activation. We therefore preexposed the cells to patients' (or control) serum at room temperature for just 30 minutes (rather than 1 hour at 4°C), to allow some reduction of AQP4 antigen density by antigenic modulation, before adding complement. We then incubated with complement at 37°C for only 30 minutes before harvesting for analysis of green and red fluorescence intensities. Exposure to active or inactive complement had no effect on either AQP4+ or AQP4− cells in the presence of control patients' serum (figure 4, A and B). NMO patients' serum had no effect on membrane integrity when complement was inactivated, regardless of AQP4 expression in the target cells. However, with active complement there was an average 5.6-fold (560%) increase in the membrane permeability of AQP4+ cells compared with AQP4− cells (figure 4B). Thus, the cytotoxicity of NMO patients' serum depended on surface expression of AQP4 and on the availability of active complement.
AQP4-specific autoantibodies are not IgM class.
Vasculocentric deposits of IgG, IgM, and complement are a hallmark of histopathological lesions found in CNS tissue of NMO patients.9,14,15 Because pentameric IgM has five Fc effector domains, it activates the complement cascade more efficiently than does IgG (a single Fc domain). Therefore, we analyzed sera and CSF of NMO patients for AQP4-specific IgM. By indirect immunofluorescence evaluation on sections of AQP4-rich mouse tissues,1 none of 31 individual NMO patients' sera or of 28 NMO patients' CSF specimens had detectable AQP4-specific IgM (data not shown). We used the flow cytometry assay to test for functional evidence of a complement-activating, AQP4-specific IgM in serum from patients who were either positive or negative for AQP4 antibody of IgG class in the course of clinical immunofluorescence evaluation.1 Only the AQP4 IgG-positive sera (pools 1–4) were cytotoxic for AQP4+ cells. This cytotoxic activity was lost when IgG was depleted (figure 4C). Recovery of AQP4-specific complement activation restored by IgG from the original positive serum pools was proportional to the percent total recovery of IgG from the protein G–agarose beads (figure 4C).
To determine immunohistochemically the frequency of IgG and IgM production in inflammatory CNS lesions of NMO patients, we investigated accumulations of differentiated plasma cells15 using fluorochrome conjugated anti-human IgG and IgM class-specific reagents at limiting dilutions to avoid interference from background extracellular IgG and IgM.9 The medullary lesion of the illustrated patient (figure 4D, left) contains abundant CD138-positive plasma cells and is in a region normally rich in AQP4.15 Plasma cells in the medullary lesion stained brightly for cytoplasmic IgG, but cytoplasmic IgM was not detected in any plasma cell (figure 4D, middle). We confirmed the immunoglobulin class specificity of our detection antibody reagents on control sections of human lymph node tissue. Clusters of plasma cells exhibited bright, cytoplasmic IgG, and scattered plasma cells exhibited bright, cytoplasmic IgM (figure 4D, right); very few cells were dually labeled (not shown). In summary, these data suggest that the IgM deposited with IgG and complement products in AQP4-rich regions of NMO patients' CNS tissues9,14,15 is not AQP4 specific.
AQP4-enriched astrocytic processes surround nodes of Ranvier in spinal cord and optic nerve.
Involvement of myelin in the pathology of NMO remains to be explained in the context of AQP4. Freeze-fracture studies in the early 1970s defined assemblies of particles as an ultrastructural characteristic of astrocytic endfeet surrounding axons at the nodes of Ranvier.21 These assemblies correspond to recently identified orthogonal arrays formed by AQP4 homotetramers in transfected cells.16 We analyzed the proximity of AQP4-specific immunoreactivity in astrocytic membranes to the nodes of Ranvier by examining sections of mouse spinal cord and optic nerve tissues by confocal fluorescence microscopy. We used immune rabbit IgG (affinity purified on AQP4 peptide) rather than NMO patients' serum because the latter is a complex mixture of multiple autoantibodies8,22 and is not amenable to affinity purification, because it binds only to native AQP4 and not to denatured, membrane-extracted AQP4 or synthetic AQP4 polypeptides (V.A. Lennon and T.J. Kryzer, unpublished observations). AQP4 was localized with respect to nodal regions by simultaneously immunostaining sodium channels,23 which are densely concentrated in the nodal axonal membrane (or Caspr1, an abundant paranodal protein24) and glial fibrillary acidic protein (GFAP), the cytoplasmic intermediate filament of astrocytic processes.25 In both the spinal cord and optic nerve, AQP4 immunoreactivity was intense near astrocytic elements and encircled periodic axonal segments expressing sodium channel immunoreactivity (figure 5A). The concentric paranodal AQP4 immunoreactivity was brighter than the background mesh pattern of astrocytic AQP4 characteristic of CNS white matter1 and was devoid of GFAP, which astrocytic foot processes lack.15 As shown in figure 5A, the extracellular space surrounding astrocytic processes is enlarged in the region of the axon segment bearing sodium channels.23 The juxtaposition of intense AQP4 and Caspr1 immunoreactivities (figure 5B) is consistent with extension of the astrocytic foot process beyond the node of Ranvier into the paranode.
Our results support a role for AQP4-specific IgG (predominantly IgG1), but not IgM, as a pathophysiological effector in the CNS inflammatory disease of patients harboring this autoantibody. In our clinical experience, it is uncommon to find NMO-IgG restricted to CSF. This observation, and the infrequency of oligoclonal IgG banding in CSF of NMO patients (by comparison with MS),8 suggest that pathogenic NMO-IgG is synthesized in peripheral lymphoid tissues rather than in the CNS compartment. If this IgG were to contact astrocytic endfeet, even in small quantities, the outcomes we demonstrated after its binding to AQP4's extracellular domain could impact astroglial polarity and compromise endfoot membrane integrity, osmotic regulation, blood–brain barrier function, and maintenance of paranodal and synaptic microenvironments. The rapid endocytosis and degradation of surface AQP4 induced by IgG binding suggest a physiologic substrate for two recently described findings in CNS tissues of patients with NMO: 1) loss of AQP4 observed immunohistochemically in putative early lesions that are inflammatory but lack demyelination,15 and 2) reversible, NMO-typical radiologic lesions observed in the area postrema and in other circumventricular regions of high AQP4 expression.13 Rapid replenishment of the plasma membrane by newly synthesized AQP4, which our study demonstrated after removal of NMO-IgG from the extracellular environment, supports the potential for a reversible insult, at least in the early stages of the disease process. This observation plausibly explains the prompt reversal of neurologic impairment reported in some patients after early plasma exchange.10
Our in vitro observations predict that timely availability of complement components at sites of IgG interaction with AQP4 (i.e., before downregulation of surface AQP4 expression) would trigger an explosive inflammatory cascade. The relative predominance of antigen modulation and complement activation, which represent competing sequelae of IgG1 binding to surface AQP4, may be critical determinants of an individual patient's clinical presentation, response to therapy, and disease course. The extensive array of Fc domains presented by IgG bound to complementary homotetramers of AQP4 in astrocytic endfeet would fulfill the precise geometric alignment required to accommodate the requisite first component of the classic complement cascade, C1q.26 This array of epitopes is analogous to the close packing of nicotinic acetylcholine receptors in the postsynaptic membrane of skeletal muscle that enables explosive activation of complement in the acute passive transfer experimental model of myasthenia gravis.27 Astrocytes likely contribute actively to inflammatory sequelae in NMO, because they produce and respond to proinflammatory cytokines and can produce and secrete all components of the classic and alternative complement systems.28 Their own membranes are relatively resistant to complement lysis because of their endowment with complement-regulatory proteins,28 but, regardless of membrane lysis or myelin loss,15 activation of early components of the complement cascade on astrocytic foot processes would initiate the intense infiltration of inflammatory leukocytes that is characteristic of NMO lesions.9,14,15
Complement fragments generated in this process would enhance endothelial permeability and promote focal parenchymal influx of serum immunoglobulins. IgMs with rheumatoid factor-like capacity to bind to Fc domains of antigen-bound IgG would amplify inflammation at astrocytic endfeet targeted by AQP4-specific IgG, thus perpetuating complement activation. This suggestion is supported by reports that serum IgM levels are frequently elevated in patients with the lupus-related anti-Ro (SS-A) antinuclear antibody,29 which is found in 16% of patients with NMO.22
Several plausible mechanisms might initiate demyelination in NMO. Firstly, oligodendrocytes are more susceptible than astrocytes to lethal injury by noxious stimuli.30 Injury to the oligodendrocyte that produces and maintains myelin would be anticipated at paranodes where they directly contact AQP4-containing astrocytic foot processes. Alternatively, secondary demyelination might result from axonal injury attributable to alteration of the ionic microenvironment at the internode. Glutamate toxicity is a third potential mechanism for initiating demyelination. The astrocytic glutamate transporter, GLT1 (also known as EAAT2), which is critical for clearing glutamate from excitatory synapses, depends on AQP4 expression for its own expression.31 Thus, a regional increase in glutamate levels, resulting from the downregulation of astrocytic AQP4 by NMO-IgG, may kill oligodendrocytes that express calcium-permeable glutamate receptors in their processes.30,32 Therapeutic blockade of AMPA receptors was shown to ameliorate neurologic sequelae in animal models of MS.33,34 Clinical trials of glutamate receptor blockade are warranted to investigate its effectiveness as a therapeutic strategy for protecting myelin and axons in acute NMO.
The authors thank Denice Bredlow, Aaron Maixner, and members of the Neuroimmunology Laboratory and the Mayo Flow Cytometry/Optical Morphology Core Facility for technical assistance, and Dr. John Henley for suggesting use of the EEA1 marker of endolysosomal targeting of IgG cross-linked AQP4.
Supplemental data at www.neurology.org
e-Pub ahead of print on October 10, 2007, at www.neurology.org.
This work was supported by the Mayo Foundation and a grant from the Ralph Wilson Medical Research Foundation.
Disclosure: In accordance with the Bayh–Dole Act of 1980 and Mayo Foundation policy, Drs. Lennon and Lucchinetti and Mr. Kryzer stand to receive royalties for intellectual property related to the AQP4 autoantigen. This intellectual property is licensed to a commercial entity for development of a simple antigen-specific assay to be made available worldwide for patient care. The test will not be exclusive to Mayo Clinic. To date, the authors have received a total of <$10,000 in royalties. Mayo Clinic offers the test as an indirect immunofluorescence assay to aid the diagnosis of neuromyelitis optica, but the authors do not benefit personally from the performance of the test. The other authors report no conflicts of interest.
Presented by Dr. Vanda Lennon as a "Frontiers in Neuroscience" Lecture at the 58th Annual Meeting of the American Association of Neurologists, San Diego, CA, April 6, 2006.
Received June 13, 2007. Accepted in final form September 4, 2007.
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Lennon VA, Kryzer TJ, Pittock SJ, Verkman AS, Hinson SR. IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J Exp Med 2005;202:473–477.
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Jarius S, Franciotta D, Bergamaschi R, et al. NMO-IgG in the diagnosis of neuromyelitis optica. Neurology 2007;68:1076–1077.
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Wingerchuk DM, Hogancamp WF, O'Brien PC, Weinshenker BG. The clinical course of neuromyelitis optica (Devic's syndrome). Neurology 1999;53:1107–1114.
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Lucchinetti CF, Mandler RN, McGavern D, et al. A role for humoral mechanisms in the pathogenesis of Devic's neuromyelitis optica. Brain 2002;125:1450–1461.
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Keegan M, Pineda AA, McClelland RL, Darby CH, Rodriguez M, Weinshenker BG. Plasma exchange for severe attacks of CNS demyelination: predictors of response. Neurology 2002;58:143–146.
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Cree BA, Lamb S, Morgan K, Chen A, Waubant E, Genain C. An open label study of the effects of rituximab in neuromyelitis optica. Neurology 2005;64:1270–1272.
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Roemer SF, Parisi JE, Lennon VA, et al. Pattern-specific loss of aquaporin 4 immunoreactivity distinguishes neuromyelitis optica from multiple sclerosis. Brain 2007;130:1194–1205.
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Rasband MN, Peles E, Trimmer JS, Levinson SR, Lux SE, Shrager P. Dependence of nodal sodium channel clustering on paranodal axoglial contact in the developing CNS. J Neurosci 1999;19:7516–7528.
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- 34.
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