text:
<?php namespace Anomaly\UsersModule\Http\Middleware;

use Anomaly\Streams\Platform\Message\MessageBag;
use Anomaly\Streams\Platform\Support\Authorizer;
use Anomaly\UsersModule\User\Contract\UserInterface;
use Closure;
use Illuminate\Contracts\Auth\Guard;
use Illuminate\Http\Request;
use Illuminate\Routing\Redirector;
use Illuminate\Routing\Route;
use Illuminate\Session\Store;

/**
 * Class AuthorizeRoutePermission
 *
 * @link          http://pyrocms.com/
 * @author        PyroCMS, Inc. <support@pyrocms.com>
 * @author        Ryan Thompson <ryan@pyrocms.com>
 */
class AuthorizeRoutePermission
{

    /**
     * The auth guard.
     *
     * @var Guard
     */
    protected $auth;

    /**
     * The route object.
     *
     * @var Route
     */
    protected $route;

    /**
     * The session store.
     *
     * @var Store
     */
    protected $session;

    /**
     * The redirect utility.
     *
     * @var Redirector
     */
    protected $redirect;

    /**
     * The message bag.
     *
     * @var MessageBag
     */
    protected $messages;

    /**
     * The authorizer utility.
     *
     * @var Authorizer
     */
    protected $authorizer;

    /**
     * Create a new AuthorizeModuleAccess instance.
     *
     * @param Guard $auth
     * @param Route $route
     * @param Store $session
     * @param Redirector $redirect
     * @param MessageBag $messages
     * @param Authorizer $authorizer
     */
    public function __construct(
        Guard $auth,
        Route $route,
        Store $session,
        Redirector $redirect,
        MessageBag $messages,
        Authorizer $authorizer
    ) {
        $this->auth       = $auth;
        $this->route      = $route;
        $this->session    = $session;
        $this->redirect   = $redirect;
        $this->messages   = $messages;
        $this->authorizer = $authorizer;
    }

    /**
     * Check the authorization of module access.
     *
     * @param  Request $request
     * @param  \Closure $next
     * @return \Illuminate\Http\RedirectResponse
     */
    public function handle(Request $request, Closure $next)
    {
        if (in_array($request->path(), ['admin/login', 'admin/logout'])) {
            return $next($request);
        }

        if ($request->segment(1) == 'admin' && !$this->authorizer->authorize(
                'streams::general.control_panel'
            )
        ) {
            abort(403);
        }

        /**
         * Check if the user is an admin.
         *
         * @var UserInterface $user
         */
        if (($user = $this->auth->user()) && $user->isAdmin()) {
            return $next($request);
        }

        $permission = (array)array_get($this->route->getAction(), 'anomaly.module.users::permission');

        if ($permission && !$this->authorizer->authorizeAny($permission, null, true)) {

            $redirect = array_get($this->route->getAction(), 'anomaly.module.users::redirect');
            $intended = array_get($this->route->getAction(), 'anomaly.module.users::intended');
            $message  = array_get($this->route->getAction(), 'anomaly.module.users::message');

            if ($message) {
                $this->messages->error($message);
            }

            if ($intended !== false) {
                $this->session->put('url.intended', $request->fullUrl());
            }

            if ($redirect) {
                return $this->redirect->to($redirect);
            }

            $route = array_get($this->route->getAction(), 'anomaly.module.users::route');

            if ($route) {
                return $this->redirect->route($route);
            }

            abort(403);
        }

        return $next($request);
    }
}

redpajama_set_name: RedPajamaGithub


Ashley Herrman, MPH, starts with the Coalition on August 20, 2018. She will be working in a position that is new to the Coalition, the Community Outreach and Project Development Director, which has lead responsibility for broadening the Coalition statewide network. Ashley has a wealth of knowledge and experience. She has a background in public health with a Masters of Public Health from the University of Missouri Columbia and a Bachelor's of Science in Health Science from Truman State University. Ashley has worked in local public health agencies as well as in non-profit organizations. She has extensive experience in tobacco control and prevention, as well as other healthy living initiatives such as access to healthy food and increasing community health through physical activity. Ashley is an advocate for oral health and community relationship building to advance public health policies both locally and at a state level.


Nevada Gov. Brian Sandoval reportedly made his case to keep online gambling legal in a meeting this week with US Attorney General Jeff Sessions. He also told the DOJ's head that regulation of iGaming has worked, in his state.
We learned earlier in the week that Sandoval and Sessions would be meeting. The topics to be discussed were not known.
It was a fair guess that online gambling would come up. Rumors had been swirling of late that Sessions might look to deal a blow to legal online gambling in the US.
Sandoval, for his part, is the vice chair of the National Governors Association. That group had sent a letter to Sessions to ask him to stay out of it, letting states handle regulation.
States have proven to be effective regulators of online gambling, so far, on geolocation, identity verification and prevention of play by minors. Online gambling also exists with little problem in highly regulated markets outside of the US, such as the UK.
It's not clear how, or if, Sessions reacted to the plea of Sandoval.
Nevada is one of three states where online gambling is legal. The others are New Jersey and Delaware. Other states, such as Pennsylvania, are currently considering legalizing online poker and casinos.
If Sessions were to do something about online gambling from his position, it would be difficult to see how he could do as Sandoval asks.
Sessions' impact on iGaming would be to attempt to roll back the 2011 Department of Justice memo on the Wire Act. That document encouraged states to move forward with iGaming. There's no obvious way Sessions could carve out Nevada from his seat.
The meeting came as the media has been taking a closer look at a $5 million donation from casino mogul Sheldon Adelson to the inauguration of President Donald Trump. Adelson is a proponent of a federal ban on online gambling. He is likely trying to parlay access to Trump into traction for an iGaming ban.
Next Story → Are PokerStars' Randomized Rewards Working Out So Far?


text:
South Pacific Books
Where? South Pacific Books » Pasifika » Category » Medicine
Total 293K jump to: go ‹ previous12345...next ›
A Country Nurse: From Wave Hill to Rural Queensland and Almost Everywhere in Between (Trade Paperback / Paperback)
By Hayes, Thea
In Stock At Publisher
Thea Hayes spent twenty years living and working on Wave Hill Station in the Northern Territory. She arrived as a naive nineteen-year-old trainee nurse from Sydney, but when she left in 1979 she was married with four children and eager for her next adventure. And what twists and ...turns her new life in rural Queensland had in store. From a stint running a corner shop in the small town of Toogoolawah to dairy and cattle farming and working as a nurse in hospitals and nursing homes, Thea's life was eternally colourful. At the age of sixty-five, after losing her beloved husband Ralph, Thea moved to London to work as a nurse and travel around Europe. Back home in Australia, she found a second chance at love with a country boy from WA, and her new life with Bob began with a caravan, a dangerous farming floodplain and a swag full of laughs. A Country Nurse charts Thea's rich and inspiring life, from Wave Hill to North Stradbroke Island; London to the Riverina in NSW, and just about everywhere in between. This is the story of an ordinary girl from Sydney who has lived her extraordinary life to the fullest.
Published AU
In stock at publisher; ships 6-12 working days
View details for this title
Australia's War Against Rabbits: The Story of Rabbit Haemorrhagic Disease (Paperback)
By Cooke, Brian Douglas
Available At Publisher
Provides up-to-date information on the use of biological control agents to manage wild rabbit populations.
CSIRO Publishing
Showing available at publisher; usually ships 7-14 working days
Herbs: An A-Z Guide to Gardening, Cooking & Health (Paperback)
By Reader's Digest
Imp. $47.99
Not Available Locally
Herbs are the most hardworking of plants. They have been used for thousands of years to flavour and preserve food, treat ailments, ward off pests and diseases, freshen the air, and decorate and enhance our lives. Over the centuries, they have also become associated with fascinati...ng myths and folklore. This practical directory provides information on how to grow, harvest and store herbs and how to make the best use of them in your daily life. You'll find recipes, homemade beauty products, safe herbal remedies and ways to make and use herbal cleaning products in the home. * An A-Z guide to growing, harvesting, storing and using more than 100 herbs. * More than 50 recipes for easy cooking. * Safe herbal treatments for common health conditions. * Save money with natural beauty products and herbal household cleaners. * More than 400 full-colour photographs.
Reader's Digest (Australia) Pt
Internationally sourced on backorder; allow 4-8 weeks
Evidence-Based Practice Across the Health Professions (Paperback, 2nd Revised edition)
By Hoffmann, Tammy; Bennett, Sally, MS; Del Mar, Christopher
Provides the reader with a foundation in the knowledge and skills necessary to perform and understand the implications of evidence-based practice within a healthcare environment. This title includes a range of practical strategies to facilitate shared decision making and effectiv...e communication with clients for improved client outcomes.
this title has been replaced by: 9780729542555
Elsevier Australia
Showing available from publisher, allow 2-4 weeks for delivery
Anxiety: Expert Advice from a Neurotic Shrink Who's Lived with Anxiety All His Life (Trade Paperback / Paperback)
By Cross, Dr Mark
Expert advice from a neurotic shrink who's lived with it all his life In his trademark warm and friendly style, Dr Mark Cross, the author of CHANGING MINDS (2016), demystifies this mental illness that affects up to one in four Australians (one in three women, one in five men), an...d how it can impact at different life stages. He looks at treatments, both medical and natural, and offers his own experiences as well as stories from others to help sufferers lead great lives while living with anxiety.
ABC Books
Available for pre-order, ships once released
(Pre-order) Add to Basket
A Beginner's Guide to Essential Oils: Hachette Healthy Living (Trade Paperback / Paperback)
By Butterworth, Lisa
Need more energy before you exercise? Want to get the most out of your training? Looking for an easy, nutritious solution? Sports smoothies are the answer. In this fully illustrated guide, each recipe is designed with the right balance of easy-to-digest carbohydrates, healthy fat...s and proteins to deliver the nutrients your body needs at each stage of training. From pre-workout smoothies to hydrate and energise, to post-workout smoothies for recovery and repair - plus muscle-building and carb-loading smoothies - you'll love how effective and delicious they are, no matter whether you're exercising day-to-day or training for a big event.
Can You Die of a Broken Heart?: A Heart Surgeon's Insight into Keeping Your Most Vital Organ Healthy (Paperback)
By Stamp, Nikki
'Dr Stamp is so clearly in love with her subject: that wonderful and yet still mysterious organ, the human heart.' Michael Mosley. When actress Debbie Reynolds died a day after her beloved daughter, Carrie Fisher, the world diagnosed it as 'heartbreak'. But what's the evidence? D...oes emotional upheaval affect the heart? Can love, or chocolate, really heal our heart problems? And why do we know so much about heart attacks in men, when they are more fatal in women? Heart and lung surgeon Dr Nikki Stamp takes us into the operating theatre, explaining what she sees in patients with heart complications and how a life-saving transplant works. Stamp fell in the love with the heart as a child and continues to be fascinated by its workings and the whole-of-life experiences that affect it. Rich with anecdotes, and insights for maintaining heart health, Can You Die of a Broken Heart? is the international bestseller from a uniquely positioned young specialist.
Better Sleep Sooner: Turn Your Sleeplessness into Sleeping Bliss, Naturally (Trade Paperback / Paperback)
The Better Sleep Sooner strategies will change the way you think about sleep, the way you go to sleep and most importantly, the way you feel when you wake up in the morning.
Aaron McLoughlin
Available at publisher; ships 6-14 working days
High Risk: A doctor's notes on pregnancy, birth, and the unexpected (Trade Paperback / Paperback)
By Karkowsky, Chavi Eve
One doctor's testament to the importance of listening - truly listening - to women and their medical experiences of pregnancy and childbirth. Although there are countless books aimed at expectant mothers and fathers, on subjects from prenatal nutrition to birthing methods, there ...is a lack of works that attempt to illuminate, unpack, and chronicle the complex, potentially life-and-death truth of this fundamental human experience. High Risk fills that gap. From an expert in maternal-foetal medicine - the subspecialty of obstetrics that concerns high-risk pregnancies - here is a timely and unflinching account of all that pregnancy, labour, and birth can entail. As an outspoken champion of women's health, a mother of four, and a humane, wry, and altogether remarkable writer, Dr Chavi Eve Karkowsky tells of miracles and joy, but also of challenge and loss - illustrating the complexity of reproduction and the health services that surround it. With historical insight and journalistic verve, she unpacks what is involved for women, for a family, and for us as a society, and explores what's at risk when these aspects of life remain clouded in mystery and misinformation.
Scribe Publications
Whaiora: Maori Health Development (Paperback, 2nd Revised edition)
By Durie, Mason
This work documents progress in Maori health development over the 20th century with a special emphasis on the post-1980s. This second edition updates the first by five years and takes account of the changing government objectives for Maori health and Maori priorities for health d...evelopment.
Oxford University Press (S2)
School & Public Library Accounts - please log in
Activities & GamesChildren's and EducationalFiction (Child / Teen)First ReadersHeinemann-RaintreeLanguage & LiteratureLearning ResourcesNon-Fiction (Child / Teen)Picture BooksPlayscripts (Kids / Teens)Reference

redpajama_set_name: RedPajamaCommonCrawl


We operate an email mailing list program, used to inform subscribers about products, services and/or news we supply/publish. Users can subscribe through an online automated process where they have given their explicit permission. Subscriber personal details are collected, processed, managed and stored in accordance with the regulations named in 'The policy' above. Subscribers can unsubscribe at any time through an automated online service, or if not available, other means as detailed in the footer of sent marketing messages. The type and content of newsletter messages subscribers receive, and if it may contain third party content, is clearly outlined at the point of subscription.
Email newsletter messages may contain tracking beacons / tracked clickable links or similar server technologies in order to track subscriber activity within email newsletter messages. Where used, such newsletter messages may record a range of subscriber data relating to engagement, geographic, demographics and already stored subscriber data.
Error: Error validating access token: Session has expired on Tuesday, 12-Mar-19 05:53:38 PDT. The current time is Friday, 19-Apr-19 17:25:51 PDT.


Using food rewards to train your dog or puppy is a very effective way to get results quickly. However, its important to use food rewards properly and effectively.
A dog's adolescence is the time when everything starts to fall apart, unless you make a concerted effort to see it through to the stability of adulthood. Your dog's adolescence is a critical time. If you ignore your dog's education now, you will soon find yourself living with an ill-mannered, under-socialized, hyperactive animal.
Here are some things to watch for.
s, heartworm prevention, toys, training, and food can add up to $680 or more. Throw in routine expenses such as dental care ($40 to $80 per year), food ($240 per year), and grooming ($30 per visit) and you're looking at $300-$400 per year before major medical expenses.


Should Sharon And Rey To Fall In Love On The Young and the Restless?
Would It Be 'Rash' For Sharon And Rey To Fall In Love On The Young and the Restless?
Sharon and Rey are most definitely not a couple in the making on The Young and the Restless.
She just broke up with Nick (Joshua Morrow) and gently rebuffed Billy (Jason Thompson) when he tried to play rebound, and Rey (Jordi Vilasuso) is totes married! Mia (Noemi Gonzalez) keeps reminding Sharon (Sharon Case) of just that.
Sharon also keeps reminding Rey how much she is rooting for him and Mia to reunite. Look, here's a Top of the Towers gift card to prove it!
Meanwhile, Rey probably keeps telling himself that the only reason he even still talks to Sharon is that she is his best lead in the JT (Thad Luckinbill) investigation. Oh, and because her desk is kind of right next to his.
But they absolutely, positively, are not falling in love.
Uhm… should they? Here's how almost 9,000 viewers voted.
Watch out, Sharon, 51% are screaming, that guy may seem like he is into you, but the viper he's married to isn't your biggest liability. The man only started talking to you in the first place because he came to Genoa City with a job to do. And that job was to arrest somebody – anybody – for killing JT.
VERY rich people very rarely go to jail. So, they're coming for Sharon, first.
It was almost a tie, as 49% of you urged Sharon to go get herself a piece of that. The man is mighty fine to look at, he seems sweet and caring, plus he can change a tire and fix a sink.
And don't worry about Mia. Once she gets a better sense of just how rich – in addition to good-looking – the men of GC are, she'll be chomping at the bit to move onto greener pastures.


text:
Recruitment for the positon of Trainees
Indian Statistical Institute (ISI), Library, Documentation & Information Science Division, Kolkata is looking for young, dynamic, tech-savvy, highly motivated and committed post graduates in Museology from recognised Indian Universities with consistent good academic record to serve as Trainee in the Prasanta Chandra Mahalanobis Museum & Archives, Kolkata purely on temporary basis to work on documentation and digitization of archival documents and museum artefacts of P.C.M. Memorial Museum & Archive.
No. of position(s): Four (04)
Contact person: Prasanta Chandra Mahalanobis Museum & Archives, (033)-2575-2100
Recruitment for the position of two Project linked persons
Indian Statistical Institute (ISI), Kolkata is inviting applications for the recruitment of Two (02) Project linked persons purely on temporary basis to work in a project funded by the National Geospatial Programme (NGP) Division, Department of Science & Technology, Government of India and undertaken by the Machine Intelligence Unit of the Institute, Kolkata. The work is on developing application for Geospatial/ Spatio-temporal data analytics based on Artificial Intelligence (AI) and Machine Learning (ML).
Contact person: Machine Intelligence Unit, (033)-2575-3100
Click here to apply: https://forms.gle/EyneVDiFkdDwk4dA9
Recruitment for the positions of Trainee
Indian Statistical Institute, Documentation & Information Science Division, Kolkata is inviting applications for the recruitment of three (03) Trainees purely on temporary basis for various functions and services in the Library of the Institute, Kolkata.
Contact person: Library, (033)-2575-2100
Indian Statistical Institute (ISI), Kolkata is inviting applications for the recruitment of one (01) Project linked person purely on temporary basis to work full time in the interdisciplinary research project titled "Spatial modelling of Arsenic contamination data from West Bengal and Bangladesh" jointly at Interdisciplinary Statistical Research Unit and Geological Studies Unit of the Institute, Kolkata.
The recruitment to the posts of Assistant 'A' (Repro-Photo/Laboratory/ Library/ Printing) vide our Advertisement No.REC-9/2017-4, KOL dated- 05/09/2017 published in the Employment News Vol. XLII No.25 on 16/09/2017 stands cancelled due to administrative reasons.
Recruitment of Professor (ONE post) in the Institute for its North-East Center at Tezpur, Assam
The Indian Statistical Institute (ISI), an Institute of National Importance, invites applications from Indian Nationals who are exceptionally bright, experienced, highly motivated with an established record of involvement in high-quality research and development activity and having strong commitments to teaching as well as academic administration, for the position of Professor (ONE post) to be posted in the North-East centre of the Institute.
Corrigendum to the advertisement number No.PU/507/ADV/2222 dated 08 July 2020 of Recruitment of Professor (ONE post) in the Institute for its North-East Centre at Tezpur, Assam.
The Institute invites applications for one post of DEPUTY CHIEF EXECUTIVE (ADMINISTRATION) 'B' on deputation basis.
(1) "Robust and Minimum Divergence Statistical Inference" and (2) "Outlier-Robust Methods in Biostatistics and Bioinformatics using Density Power Divergence"
Applications are invited for recruitment of two (02) Project linked persons (one for each of) on purely temporary basis in the Interdisciplinary Statistical Research Unit of the Institute, Kolkata.
Contact Person: Interdisciplinary Statistical Research Unit, 033-2575-5054
1. Recommender System for Citations and Illustrations, 2. DADDI: Deep Analysis of Degraded Document Image, 3. DAMP: Deep Analysis for Pain Management, 4. 4.	Cyclicity Detection
Walk-in interview will be conducted on 05 August 2020 and 06 August 2020 at the Computer Vision & Pattern Recognition (CVPR) Unit Seminar Room (8th Floor, S.N. Bose Bhawan), Indian Statistical Institute, 203 B.T. Road, Kolkata - 700108 for recruitment of four (04) Project linked persons (one for each of) purely on temporary basis.
Contact Person: Computer Vision & Pattern Recognition (CVPR) Unit, 033-2575-2850
Land leasing arrangements and functions in eastern India
Applications are invited from Indian nationals for recruitment of one (01) Project linked person on purely temporary basis in the Sociological Research Unit of the Indian Statistical Institute, Giridih.
Contact Person: Sociological Research Unit, Giridih, 06532-222034
Faculty Advertisement for Reserved Categories
Paleogeography of the Neogene Foreland Basin of Eastern Himalaya and its relationship with contemporaneous sediments of Mizoram
Project Linked Person for Research Projects in Advanced Computing & Microelectronics Unit
1. Investigating Multiple Kernel Approaches for Efficient and Effective Multi-View Clustering; 2. Soccer Analytics using Computer Vision


text:
Customer Generation
Home - News - MVF Shortlisted For Drum Marketing Award
MVF Shortlisted For Drum Marketing Award
MVF and its client, Amplifon, have been shortlisted for Health Marketing Strategy of the Year in the prestigious Drum Marketing Awards.
Amplifon is a leading worldwide provider of digital and invisible hearing aids, with more than 200 hearing centres in the UK alone. Amplifon's global company objectives are to scale their operations and increase revenues.
In 2017, Amplifon partnered with MVF to see if they could reach a new market of digital users online to grow their business. What started off as a single UK campaign is now a comprehensive, multi-channel digital marketing strategy to help improve the hearing of millions of people around the globe.
MVF COO Andrew Harkness commented: "MVF focuses on understanding how best to create value for our clients, then works to realise that value and develop long-term performance based partnerships. With Amplifon, we have moved beyond being just another marketing channel, and we have created new capability that delivers sustainable growth for our client. I am so proud of the results our teams have been delivering in partnership with Amplifon, and I am excited about what we can achieve together in the future."
Amplifon Marketing Director Daniela Cantale commented: "By working in close collaboration with MVF, we have achieved our goal to share our services with a wide range of potential customers by designing highly targeted digital marketing campaigns appealing to audiences likely to convert."
MVF and Amplifon are up against health sector heavyweights BUPA and Optum in the Health Strategy category, with the winner to be announced at an awards ceremony in London on 30 May.
MVF Achieve Best Companies 3 Star Accreditation
We're extremely thrilled to have, once again, achieved a 3 Start Accreditation from the Best Companies research agency, which supplies data to the Sunday Times for its annual Best Companies to work for.
MVF Shortlisted for Two UK Biddable Media Awards
MVF's Linkedin team has been shortlisted for not one, but TWO UK Biddable Media Awards - Best Use of Linkedin Ads and Best Use of Data.
MVF Shortlisted for REBA Employee Wellbeing Awards
This week REBA announced that MVF has been selected as a finalist for Best New Wellbeing Strategy!
MVF is Recognised as a Top 10 Company to Work for in the UK by the Sunday Times
The company are celebrating after achieving 10th place in the Sunday Times 100 Best Small Companies List, meaning they are a top 5 organisation to work for in London.
MVFer Named Winner at the WeAreTheCity Rising Stars Awards
Our sales academy graduate has been named a winner for the WeAreTheCity Rising Stars in Technology award.
MVF Shortlisted for Marketing Team of the Year at the Masters of Marketing Awards
MVF's biddable team has been shortlisted for Marketing Team of the Year at the Masters of Marketing Awards.
MVF Locations
MVF UK
Imperial Works
Block C, Perren Street
London NW5 3ED
MVF US LLC
801 Barton Springs Road
MVF © 2016. All rights reserved / Privacy Policy & Legal


text:
Q: This UWP process is suspended to improve system performance ... but it isn't a UWP app We have a WPF app build on .NET Framework 4.8, but recently we saw this in the taskbar (running on windows 10 x64) :

When hovering over the WPF client, the tooltip said 'This UWP process is suspended to improve system performance'. Our app was also hanging for a while before doing some work again. How is this possible? We're not running on .NET Core 3.0, no desktop bridge, no UWP integration at all ... it's just a normal WPF app.

redpajama_set_name: RedPajamaStackExchange


text:
Q: Show the difference of lines between two files How to implement a functionality in emacs to show the difference of contents between two files opened in two buffers?
Kind of like revdiff when using mercurial.

A: BTW, if you want to see the differences between two blocks of text in the same buffer, you can do it as follows:
- make sure smerge-mode is loaded (e.g. M-x load-library RET smerge-mode RET).
- go to the beginning of the first bloc, C-SPC
- go the beginning of the second bloc, C-SPC
- go to the end of the second bloc, M-x smerge-makeup-conflict RET
- then you can use C-c ^ R or C-c ^ = to view the differences.
Note that this works better if the two blocs are pretty much consecutive.

A: Emacs has ediff, which can diff two files.  To use it, just do M-x ediff, and specify the two files you want to diff.

A: M-x diff does that.
See the docs for more: http://www.gnu.org/software/emacs/manual/html_node/emacs/Comparing-Files.html

A: I also found that compare-windows does an amazing job cycling through differences in opened buffers.


text:
T Cell Acute Lymphoblastic Leukemia as a Consequence of Thymus Autonomy
Luna Ballesteros-Arias, Joana G. Silva, Rafael A. Paiva, Belén Carbonetto, Pedro Faísca and Vera C. Martins
J Immunol February 15, 2019, 202 (4) 1137-1144; DOI: https://doi.org/10.4049/jimmunol.1801373
Luna Ballesteros-Arias
*Lymphocyte Development and Leukemogenesis Laboratory, Instituto Gulbenkian de Ciência, Fundação Calouste Gulbenkian, 2780-156 Oeiras, Portugal;
ORCID record for Luna Ballesteros-Arias
Joana G. Silva
ORCID record for Joana G. Silva
Rafael A. Paiva
ORCID record for Rafael A. Paiva
Belén Carbonetto
†Bioinformatics Unit, Instituto Gulbenkian de Ciência, Fundação Calouste Gulbenkian, 2780-156 Oeiras, Portugal; and
ORCID record for Belén Carbonetto
Pedro Faísca
‡Histopathology Unit, Instituto Gulbenkian de Ciência, Fundação Calouste Gulbenkian, 2780-156 Oeiras, Portugal
ORCID record for Pedro Faísca
Vera C. Martins
ORCID record for Vera C. Martins
PDF + SI
Thymus autonomy is the capacity of the thymus to maintain T lymphocyte development and export independently of bone marrow contribution. Prolonging thymus autonomy was shown to be permissive to the development of T cell acute lymphoblastic leukemia (T-ALL), similar to the human disease. In this study, performing thymus transplantation experiments in mice, we report that thymus autonomy can occur in several experimental conditions, and all are permissive to T-ALL. We show that wild type thymi maintain their function of T lymphocyte production upon transplantation into recipients with several genotypes (and corresponding phenotypic differences), i.e., Rag2−/−γc−/−, γc−/−, Rag2−/−IL-7rα−/−, and IL-7rα−/−. We found that the cellularity of the thymus grafts is influenced exclusively by the genotype of the host, i.e., IL-7rα−/− versus γc−/−. Nonetheless, the difference in cellularity detected in thymus autonomy bore no impact on onset, incidence, immunophenotype, or pathologic condition of T-ALL. In all tested conditions, T-ALL reached an incidence of 80%, demonstrating that thymus autonomy bears a high risk of leukemia. We also analyzed the microbiota composition of the recipients and their genetic background, but none of the differences found influenced the development of T-ALL. Taken together, our data support that IL-7 drives cellular turnover non–cell autonomously, which is required for prevention of T-ALL. We found no influence for T-ALL in the specific combination of the genotypic mutations tested (including the developmental block caused by Rag deficiency), in microbiota composition, or minor differences in the genetic background of the strains.
Normal T lymphocyte development occurs mostly in the thymus and depends on the lifelong seeding of hematopoietic progenitors that originate in the bone marrow. In the thymus, these cells commit to the T lymphocyte lineage and further develop to generate T lymphocytes, which then emigrate and integrate the diverse repertoire that surveys the periphery. The process of T lymphocyte differentiation is highly dynamic and involves cellular turnover at such levels that guarantee complete replacement of all thymocytes every 4 weeks (1). These and other aspects of thymus and thymocyte development can be, and have been, studied using thymus transplantation experiments. Classical experiments have shown that wild type thymi grafted into severe combined immunodeficient (SCID) recipients produced and exported a single cohort of donor-derived T lymphocytes. Thereafter, thymus grafts produced no more T lymphocytes (2). These studies have been extensively reproduced by other laboratories using recipients with other genetic mutations but with the same developmental block (3). The results have been interpreted to indicate that precursors in the thymus (thymocytes) do not have the capacity to self-renew, and this remained a central dogma of thymus and T lymphocyte biology for many years (4). This dogma was disproved by the work of two independent laboratories (5, 6). Performing thymus transplantation experiments, both groups have shown that thymus function can be maintained independently of bone marrow contribution, which indicates that some thymocytes are indeed capable of self-renewal (5, 6). Specifically, it was shown that in the absence of de novo colonization of the thymus by hematopoietic progenitors, the organ maintains the production and export of functional T lymphocytes (5). Furthermore, if (host) hematopoietic progenitors seeding the thymus were deficient for IL-7r, then thymus autonomy occurred from donor-derived thymocytes. This was the case in recipients deficient for common cytokine receptor γ chain (γc) and for recipients that were deficient for IL-7rα (5, 6), the two chains that together form the receptor for IL-7. Both studies have shown that the TCR repertoire generated was diverse (5, 6); however, one of the papers showed that after 10 weeks of progenitor deprivation, the thymus grafts already had some degree of clonal dominance (5).
Although the physiological significance of thymus autonomy remains elusive, it became clear that it is a tightly regulated process that must be kept inhibited under physiological conditions. Indeed, follow-up work by one of the laboratories showed that prolonging thymus autonomy was permissive to the development of T cell acute lymphoblastic leukemia (T-ALL) (7). The disease was similar to the human counterpart in immunophenotype, pathologic condition, and genetic and genomic lesions, as well as at the transcriptional level. This included gain-of-function mutations in Notch1, similar to those described in the majority of human T-ALL (8), and expression of Lmo2 and Tal1, which are common to the majority of pediatric T-ALL (9–11). Nonetheless, a review article claimed that T-ALL did not develop in 19 mice used in similar experiments and further suggested that thymus transplants could be used as a therapy for correction of immunodeficiencies caused by cell-autonomous defects in T lymphocyte development (12). This was suggestive that there are important factors that could influence leukemia, and in addition to genetic factors, the animal facilities could also be relevant. Indeed, there is extensive work on the functional relevance of the composition of gut microbiota for immune function, and that identical mice from different sources, i.e., purchased from different suppliers or kept in distinct facilities, can differ in immune function (13–15).
In this study, in a new laboratory, we sought to test whether development of T-ALL from autonomy could be reproduced using newly established animal colonies, thereby excluding potential effects of the animal house on T-ALL. We covered most genotypes used by both groups, and tested whether thymus autonomy could be influenced by the genotype of the hematopoietic progenitors seeding the thymus. Furthermore, we tested whether the genotype of the recipients could impact on the development of T-ALL from wild type thymocytes, on the immunophenotype and/or severity of disease. With that purpose, we transplanted wild type thymi under the kidney capsule of recipients with the following genotypes: Rag2−/−γc−/−, γc−/−, Rag2−/−IL-7rα−/−, and IL-7rα−/−. They all have in common that they cannot respond to IL-7 but differ in phenotype and/or in their capacity to respond to additional cytokines. These recipients further differed among themselves in microbiota composition and, to some extent, in genetic background (determined as the percentage of C57BL/6J [B6] background). There were differences in the cellularity of thymus grafts in γc−/− versus IL-7rα−/− hosts during thymus autonomy. Nevertheless, all conditions tested were permissive to autonomy and subsequent development of T-ALL. We could detect no difference in the onset, incidence, pathologic condition, or immunophenotype of T-ALL between all tested conditions.
B6 (CD45.2+) mice were bred and kept at the Instituto Gulbenkian de Ciência (IGC) in a colony that is frequently replenished by mice purchased from Charles River Laboratories. B6.SJL-Ptprca Pep3b/BoyJ (CD45.1+) mice, stock no. 002014, in this article termed B6/Ly5.1, γc−/− (16), stock no. 003174, and IL-7rα−/− (17), stock no. 002295, were purchased from The Jackson Laboratory. The strain IL-7rα−/− was also crossed to Rag2−/− (18) animals (originally purchased from Taconic Biosciences and kept in a colony at the IGC) to obtain the strain Rag2−/−IL-7rα−/−. Rag2−/−γc−/− mice used in this study were imported from the University of Ulm, Germany, through embryo rederivation. Thymi for thymus transplants were harvested from F1(B6xB6/Ly5.1) newborn donors. All mice were bred and kept in individually ventilated cages in the specific pathogen–free area of the mouse facility of the IGC. All animal experiments were approved by the Ethics Committee of the IGC – Fundação Calouste Gulbenkian and the Direção Geral de Alimentação e Veterinária.
Thymus transplants
Thymi were isolated from F1(B6xB6/Ly5.1) newborn mice and grafted under the kidney capsule of recipient mice as in previous studies (5, 7). Briefly, the thymus lobes were physically separated and kept in cold PBS. Recipient mice were anesthetized with ketamine (100 mg/kg) and xylazine (16 mg/kg), and each host received one thymus, with each lobe placed in one extremity of the kidney.
T-ALL detection
Recipients were periodically bled from the submandibular vein from 16 wk posttransplant onwards, and the peripheral blood was analyzed for the presence of T lymphocytes and/or blasts. All animals showing clinical signs of disease and/or presence of lymphoblasts in the blood were euthanized and analyzed.
Organs were harvested and single-cell suspensions prepared in PBS/FBS. Cells were blocked with 114 μg/ml mouse IgG (Jackson ImmunoResearch) for 15 min and stained for 30 min in an appropriate diluted Ab staining solution. Second-step stainings were performed whenever necessary, for 30 min, after washing twice. Abs and streptavidin were purchased from BioLegend and were as follows: CD3ε biotin (145-2C11), CD3ε APC-Cy7 (145-2C11), CD4 PE (GK1.5), CD4 PE-Cy7 (GK1.5), CD8a FITC (YTS169.4), CD11b biotin (M1/70), CD11c biotin (N418), CD19 biotin (6D5), CD25 BV605 (PC61), CD44-PerCP-Cy5.5 (IM7), CD45.1 biotin (A20), CD45.1 PE-Cy7 (A20), CD45.2 PerCP-Cy5.5 (104) and CD45.2 PE (104), CD45.2 PB (104), CD117 APC (2B8), CD127 APC (A7R34), γδTCR PE (GL3), Gr-1 biotin (RB6-8C5), NK1.1 biotin (PK136), Ter119 biotin (TER-119), streptavidin BV605, and streptavidin BV785. Double-negative (DN) thymocytes were defined as CD4 negative, CD8 negative, lineage negative (lineage-positive cells were defined as positive for an Ab mixture, including CD3ε, CD11b, CD11c, CD19, Gr-1, NK1.1, and Ter119). Dead cells were excluded with SYTOX Blue (Molecular Probes), Zombie APC-Cy7, or Zombie Pacific Orange (BioLegend). Sample acquisition was performed in a BD LSRFortessa X-20 cell analyzer using BD FACSDiva 8 software, and analyses were done using FlowJo.
Samples were fixed by immersion in 10% buffered formalin for ∼48 h at room temperature, with exception of the brain (1 wk). Bone samples were decalcified with an EDTA solution changed daily during 1 wk. Samples were routinely processed, embedded and sectioned into 3-μm-thick sections, and stained with H&E. Liver, brain, and male reproductive organs were submitted to gross examination and trimming to maximize the area of analysis. The liver lateral left lobe was macroscopically sectioned in four to five transverse fragments. The brain was macroscopically sectioned in four to five consecutive coronal sections, ∼2.5 mm apart, starting in the middle of the olfactory bulbs. All histology was performed by the Histopathology Unit at the IGC. Slides were analyzed with a DMLB2 microscope (Leica), and images were acquired with a DFC320 camera (Leica) and NanoZoomer-SQ Digital slide scanner (Hamamatsu Photonics).
Statistical analysis of the results was conducted in Prism 7. The survival curves in Fig. 2A were compared using a log-rank (Mantel–Cox) test, and no statistically significant difference between the curves was found. Sample groups in Fig. 2C were subjected to the D'Agostino and Pearson normality test, revealing a nonparametric distribution. Samples were compared with a Kruskal–Wallis test followed by a Dunn multiple comparison post hoc test, and no statistically significant difference was found.
Analysis of gut microbiota
Fresh fecal pellets were collected from five to seven cages per genotype, with the aim of representing the whole colony (avoiding sampling cages that came from the same breeders). DNA extraction from the mouse fecal content was performed using a QIAamp DNA Stool Mini Kit (Qiagen), according to the manufacturer's instructions. Amplification and sequencing of the 16S rRNA gene was carried out at the IGC's Genomics Unit using primers specific to the V4 region of 16S rRNA, as described (19), and paired-end sequencing (2 × 250 bp) on an Illumina MiSeq Benchtop Sequencer following Illumina recommendations.
Bioinformatic processing of raw demultiplexed reads was done using QIIME2 v.2018.4 (https://qiime2.org) with default parameters. DADA2 was used for quality filtering, denoising, paired-end merging, and amplicon sequence variant (ASV, i.e., sub–Operational Taxonomic Units) calling using qiime dada2 denoise-paired method (20). A threshold was defined at a Phred quality score of 20 for trimming before merging (parameters: –p-trunc-len-f and –p-trunc-len-r), defining 235-bp length for forward reads and 220 bp for reverse reads. After filtering, average sample size was 32,576 reads (minimum: 12,245, maximum: 62,457), and 627 ASVs were detected. ASVs were aligned using the qiime alignment mafft method (21). The alignment was used to calculate the phylogenetic distances between ASVs using qiime phylogeny fasttree (22). ASV tables were subsampled without replacement to even sample size for diversity analysis using qiime diversity core-metrics-phylogenetic pipeline. The smallest sample size was chosen for subsampling. Unweighted and weighted UniFrac distances were calculated to compare community structure (23). Taxonomic assignment of ASVs was performed using a Bayesian classifier trained with the Greengenes database (i.e., 16S rRNA sequences aligned at 99% and truncated at positions 515–806) using the qiime feature-classifier classify-sklearn method (24). UniFrac distance matrices and ASV tables were used to calculate principal coordinates and construct ordination plots using R software package version 3.4.3 (http://www.R-project.org). The significance of groups in community structure was tested using PERMANOVA. BiodiversityR version 2.8-4 and vegan version 2.4-5 packages were used, and ggplot version 2.2.1 was used for plotting. Bioinformatic processing and analysis of the data were performed at the Bioinformatics Unit of the IGC.
Genetic background determination
Enrichment of B6 genomic DNA was determined by genotyping each individual mouse for 159 informative single nucleotide polymorphisms (SNPs) evenly distributed across the genome (1 SNP/9 cM on average). Genotype detection was done using the mass spectrometry–based Agena iPlex Gold technology, which allows for multiplexing of up to 40 SNPs per reaction, totalling five multiplex reactions. In brief, a set of three primers was designed for each SNP: two to be used in a regular PCR reaction to amplify the region of the SNP of interest, and the third to be used in the iPlex reaction. This is a single base extension PCR with mass-modified nucleotides, in which the primer anneals immediately before the position of the SNP. The extension product is analyzed by time of flight, and genotypes were attributed according to the m.w. difference of the extended base relatively to the primer mass.
Thymus autonomy occurs in all IL-7r–deficient recipients: Rag2−/−γc−/−, γc−/−, Rag2−/−IL-7rα−/−, and IL-7rα−/−
Thymus autonomy has been previously reported by two independent laboratories (5, 6), and extending the period of thymus autonomy led to T-ALL (7). The first two studies had relatively short observation times posttransplantation, and only one of the groups referred to a small number of recipients that were followed for 7 mo (6). The reduced number of mice followed for that time could account for failure in the detection of leukemia. However, a more recent article refers to one cohort of Rag2−/−γc−/−–transplanted mice that was followed for 9 mo posttransplantation by the same group (12). The genotype of the recipients used for the studies on thymus autonomy did not fully overlap between the laboratories (Table I), and we reasoned that this could be one aspect impacting on autonomy. To address this point, we transplanted wild type thymi into the following recipients: Rag2−/−γc−/−, γc−/−, Rag2−/−IL-7rα−/−, and IL-7rα−/−. The cells in γc−/− mice cannot respond to IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. In IL-7rα−/− mice, cells cannot respond to IL-7 and to thymic stromal lymphopoietin. In the thymus, both γc−/− (16, 25) and IL-7rα−/− (17) mice have marked reduced cellularity, caused by the inability of thymocytes to respond to IL-7 at the DN2 stage (CD4− CD8− double negative, CD3 negative CD25 positive, CD44 positive). As a consequence, the next stage, the DN3 (CD25 positive, CD44 negative), is severely reduced, and very few thymocytes progress through differentiation. Rag2−/−γc−/− and Rag2−/−IL-7rα−/− thymocytes have, in addition, a complete developmental block at the DN3 stage, caused by the Rag2 deficiency (18), which leads to an even more severe phenotype. Thymus grafted into these mice and analyzed 9 wk posttransplantation could maintain double-positive thymocytes of donor origin in recipients from all tested genotypes (Fig. 1A). Similar to one of the reports (5), no population of thymus-donor origin resembling the DN3, according to immunophenotype, was detected at this stage (Fig. 1B). The recipients with at least one productive thymus graft were over 80% in every group (Fig. 1C). The only difference detected was a statistically significant difference between the number of CD4+ CD8+ double-positive thymocytes of donor origin in thymi grafted into γc−/− versus IL-7rα−/− (Fig. 1D) recipients.
Table I. Summary of recipient mouse strains used in thymus transplantation experiments to study thymus autonomy and/or T-ALL
Thymus autonomy occurs upon transplantation of wild type thymus donors into recipients lacking IL-7r. Wild type newborn thymi (CD45.1+ CD45.2+) were transplanted under the kidney capsule of the indicated recipients (all CD45.2+) and analyzed by flow cytometry 9 wk posttransplantation. Males and females were used. One graft showing thymus autonomy is depicted for each of the recipients, as indicated, in (A and B). All samples shown were selected from one single experiment and are representative of the phenotype obtained at this time point of analysis. (A) Expression profile for CD4 and CD8 of the cells present in the thymus grafts. Thymocytes were pregated on live, donor cells. (B) Samples in (A) were gated for CD4 negative, CD8 negative, and lineage negative (in this article referred to as DN) and analyzed for the expression of CD117 and CD25. Numbers indicate the percentages of the corresponding gates shown (early thymic progenitors [ETP], DN2a, DN2b, DN3, DN4) in donor or host DN. Experiments including every indicated group of recipients were repeated three times. (C) Quantification of the proportion of recipient mice that were productive (black section of the bars) or exhausted (white section) 9 wk after transplant. Mice were considered productive if at least one (of two) of the thymus grafts had CD4+ CD8+ double-positive thymocytes, or exhausted if both grafts lacked CD4+ CD8+ double-positive thymocytes. Numbers on the bars correspond to the number of individual mice considered productive or exhausted. (D) The number of CD4+ CD8+ double-positive thymocytes of donor origin in productive thymus grafts is depicted. Every circle corresponds to a thymus graft, and the horizontal line represents the mean. ***p = 0.0260.
T-ALL develops in Rag2−/−γc−/−, γc−/−, Rag2−/−IL-7rα−/−, and IL-7rα−/− recipients
Even if no major differences were detected 9 wk posttransplantation, it could still be that there were differences in the development of T-ALL. Therefore, we used the same transplantation setting and extended the time of observation following thymus transplantation to 70 wk posttransplantation. T-ALL developed with onset at 15–17 wk posttransplantation and reached an incidence of 74–85% in all recipients (Fig. 2A). No statistical difference was found between groups. Blast cells were detected in the blood of leukemic animals (data not shown). Organs of mice with T-ALL were analyzed by flow cytometry (bone marrow, grafted and endogenous thymi, and spleen) and were mostly composed of leukemic blast cells that had an immature surface phenotype (Fig. 2B). Although there was some variability in the immunophenotype of the different leukemias, they were most commonly immature single positive to (CD4 and CD8) double positive. In line with one previous report (7), animals had large thymus grafts and developed hepatosplenomegaly (Fig. 2C). H&E–stained sections show multiorgan neoplastic cell infiltration of monomorphous medium- to large-sized neoplastic lymphocytes, with scant cytoplasm and irregular-edged nuclei with coarsely clumped chromatin, and with numerous mitotic figures (Fig. 3A). The heart was often not affected, but when it was, it exhibited multifocal, perivascular to interstitial neoplastic lymphoid cell infiltrates. The lungs presented predominantly perivascular and peribronchial infiltrations. The liver showed perivascular and periportal cell infiltrations with invasion into the adjacent liver parenchyma. The kidney exhibited interstitial cell infiltrations, denser in the renal cortex, with invasion of the retroperitoneal and perirenal adipose tissue. The brain displayed, in most cases, a multifocal cell infiltration in the subarachnoid and pia mater and in the choroid plexus in the lateral and third ventricles. Less frequently, neoplastic cells were also identified in the olfactory bulbs and in the median eminence of the hypothalamus (Fig. 3A). The spleen showed total obliteration of the normal architecture, with white and red pulp structure loss, due to the proliferation of lymphoid cells. The bone marrow was replaced by a diffuse monomorphous neoplastic lymphoid population. In male mice, both testis and accessory glands presented an interstitial cell infiltration, whereas in female mice, there was total obliteration of the ovaries and uterus architecture due to the neoplastic diffuse cell infiltrates (Fig. 3B). Those mice that did not develop T-ALL were analyzed at the end of the experiment and, out of 15 recipient mice, across all genotypes, only one still had productive thymus grafts (had CD4+ CD8+ double-positive thymocytes of donor origin), whereas all others were exhausted, showing no sign of thymopoiesis of donor origin. This might indicate that the mice that failed to develop leukemia did so mostly because autonomous thymopoiesis stopped previously or did not take place. Overall, no differences could be determined between experimental groups in type, degree of infiltration, or in the organs affected by T-ALL (Fig. 3).
T-ALL emerges as a consequence of thymus autonomy. Wild type newborn thymi were transplanted under the kidney capsule of the indicated recipients, as in Fig. 1, and followed for 70 wk. (A) Kaplan–Meier curves depict the onset and incidence of T-ALL in recipient mice of the indicated genotypes. Number of recipient mice transplanted (n) is indicated for each genotype. (B) Mice developing T-ALL were analyzed by flow cytometry, and the immunophenotype of the cells in the thymus graft and spleen is shown for CD4 and CD8 expression in live, donor-gated cells (CD45.1+ CD45.2+). T-ALLs are representative of the most common immunophenotype, although there is some interindividual variation for the expression of CD4 and CD8 between T-ALL samples (data not shown). (C) Total cellularity of the thymus grafts (to the left) and spleens (to the right) is depicted, where each dot represents one T-ALL sample and the horizontal line represents the mean. No statistical difference was observed between the groups as analyzed with a Kruskal–Wallis test, followed by a Dunn multiple comparison post hoc test.
Histological analysis of organs in mice with T-ALL. The organs of mice developing T-ALL were harvested and analyzed by histology using an H&E staining. (A) One example per recipient strain is depicted for heart, lung, liver, kidney, and brain. Control sections were taken from a wild type C57BL/6 healthy mouse. Scale bar, 200 μm in all photos of the five top lines. The scale bar in the lowest line of photos corresponds to 3 mm. (B) Summary of the histopathological analysis of the animals with T-ALL. Each circle represents one mouse, and each section corresponds to a given organ, as indicated. The presence of infiltrating leukemic cells in a given organ appears as a red section, absence as blue, and gray indicates that the organ was not analyzed histologically. Males and females were used.
Microbiota composition and the genetic background of the recipients do not influence T-ALL
Beyond the genotype of the recipients and the consequent causes in T lymphocyte development of host origin, additional factors could have influenced the difference in T-ALL between the two laboratories (7, 12). These include the health status of the animals, the sanitary conditions in which they were kept, their microbiota composition, and even the genetic background of the recipients. Of note, the studies were performed in different research institutions, i.e., one group kept their mouse colonies in the Central French breeding facilities (6, 12), the other at the University of Ulm, in Germany (5, 7). In this study, we have established the mouse colonies newly at the IGC, Portugal. Rag2–/–γc–/– were imported through embryo rederivation from Germany and correspond to the strain used in some of the previous studies (5, 7). IL-7rα–/– were imported from The Jackson Laboratory through embryo rederivation. After rederivation, IL-7rα–/– were crossed to animals from a Rag2–/– colony that was already kept at our institute to generate the strain Rag2–/−IL-7rα–/–. γc–/– were imported from The Jackson Laboratory and tested clean upon arrival and hence were not rederived. Because all animals used in this study were imported newly into a new animal facility, they inevitably differed from the animals used in former studies. In addition, gut microbiota composition could also differ between the strains used in this study as a result of rederivation (or its absence), which was involved in the import of some of the mouse strains. To determine whether the gut microbiota composition differed between the four recipient strains used in this study, we collected fecal pellets from animals of five to seven different cages per strain and sequenced the V4 region of the 16S rRNA gene. The composition of the gut microbiota was analyzed using unweighted and weighted UniFrac phylogenetic distances. Principal coordinate analyses show that the gut community of γc–/– mice differed from that of the other strains (Supplemental Fig. 1A). These results were consistent for both the unweighted UniFrac distance (PERMANOVA, p ≤ 0.05), which is based on the presence/absence of bacterial groups, and for the weighted UniFrac distance, which also considers the relative abundance of bacterial groups (PERMANOVA, p ≤ 0.05). The differences in community structure were further evidenced by analysis of taxonomic composition, depicted in this article at the level of phyla (Supplemental Fig. 1B). Specifically, the phylum Deferribacteres was absent in the gut microbiota of the strain γc−/− (Supplemental Fig. 1B). Further differences could be detected at the level of genera and include the absence of Prevotella, Odoribacter, AF12 (phylum Bacteroidetes), Sutterella, and Bilophila (phylum Proteobacteria) in the gut microbiota of γc−/−. These genera were, however, present in Rag2−/−IL-7rα−/−, IL-7rα−/−, and Rag2−/−γc−/− (Supplemental Fig. 1C). The only genotype with a clearly different microbiota composition was γc−/−, consistent with these mice being the only ones not rederived upon import into the current facility. Nevertheless, the fact that γc−/− mice developed T-ALL similarly to the other genotypes tested indicates that T-ALL was not influenced by the microbiota.
The genetic background of the recipients could also have impacted on the results. Rag2−/−γc−/− mice used by one of the groups had been crossed into a pure B6 background (12), whereas the other was crossed to B6 for fewer generations. To determine the genetic background of the strains used in this study and define the degree of genetic backcross into the B6 background, we analyzed the genomic DNA of our strains and screened for SNPs to determine the percentage of B6 background in each chromosome (Fig. 4). As predicted, every strain had one to two SNPs positive for the strain 129 and not B6, consistent with the loci that were targeted in those strains, i.e., Rag2 in chromosome 2, IL-7rα in chromosome 15, and γc in chromosome X (Fig. 4). Beyond these loci, the only strain for which the B6 background was incomplete is the Rag2−/−γc−/−, which was the same strain used in previous work (5, 7). Nevertheless, T-ALL developed identically in the four strains, three of which were in a pure B6 background. Hence, our results show that the pure B6 background of the recipients does not protect from leukemogenesis (Fig. 4).
The genetic background of the recipients does not influence T-ALL. The genomic DNA of the indicated mouse strains was tested by SNP analysis to determine the percentage of B6 background. Chromosomes are depicted in the x-axis for one mouse of the indicated strains. Percentages were calculated as the proportion of B6-positive SNPs (all homozygote) in each chromosome. A total of two to four mice per strain were analyzed, and all animals yielded identical results within strains.
In this article, we show that transplantation of wild type thymi into recipients that are Rag2−/−γc−/−, γc−/−, Rag2−/−IL-7rα−/−, or IL-7rα−/− is followed by a period of thymus autonomy, and that is permissive to malignant transformation and development of T-ALL. Although thymus autonomy was characterized by a difference in cell numbers of donor double-positive thymocytes between the thymi transplanted into γc−/− versus IL-7rα−/− hosts, no other differences were detected among recipients, including incidence and phenotype. Furthermore, no differences for T-ALL in terms of onset, incidence, pathology, or immunophenotype of T-ALL were found among the four groups. This was so despite the small difference in genetic background (percentage of B6 background) of the Rag2−/−γc−/− recipients and the variability in composition of the gut microbiota in γc−/− recipients. These data are concordant with the concept of cell competition in the regulation of thymus turnover under physiological conditions, in which "young" precursors, with a shorter time of thymus residency, lead to the clearance of the "old" precursors, with a longer dwell time in the thymus (7). Taken together, our data support that IL-7 is a limiting factor in early T lymphocyte development, which determines the capacity of the young precursors to outcompete the old, thereby promoting cellular turnover. Although the mechanism of IL-7 regulation of thymus turnover remains elusive, several hypotheses can be considered. Because IL-7 is limiting, the differential access to the cytokine could determine the outcome of the cells: survival and expansion of the young versus clearance of the old. This outcome could be reached if the young are closer to the source of IL-7 than the old precursors, or if young and old cells express different levels of the receptor. Future work will be essential in defining the molecular mechanism of cell competition and testing which of the hypotheses is correct. Furthermore, the regulation of thymus autonomy as an outcome of the competitive disadvantage of the progenitors seeding the thymus will be crucial in understanding whether any cell that enters the thymus has the potential to become capable of sustaining thymopoiesis autonomously, or if this property is acquired de novo in specific conditions. Finally, it will be interesting to identify the molecular changes occurring that cause malignant transformation of healthy, wild type thymocytes.
The difference of results between two laboratories is rather intriguing. Although both reported on thymus autonomy (5, 6), the work that followed in the Rodewald laboratory showed that autonomy is permissive to T-ALL (7). The latter finding was challenged by Rocha's group (12). This difference has important implications, as thymus transplants were proposed as a potential therapy to be used in the clinics for correction of immunodeficiencies caused by cell-autonomous defects in thymocyte development (12). Furthermore, the discrepancy suggests that there might be factors beyond the genotypes tested that can affect leukemogenesis in thymus transplantation experiments. Whereas Rocha's former study did not follow enough mice for long enough time to detect T-ALL (6), the review article refers to a group of 19 animals followed for over 6 mo (12). It would have been interesting to know whether thymopoiesis was still active in all those animals at the end of the observation period. Although the authors suggested that inefficient backcrossing of recipients onto B6 background might have triggered disease (12), we now show that the pure B6 background per se is not sufficient to prevent leukemia. Alternatively, differences in microbiota composition could account for the dissimilar results between laboratories, but we consistently observed identical T-ALL development independently of differences in the gut microbial diversity and composition of the recipient mice. Moreover, results were reproduced in a new animal facility, likely to differ significantly in gut microbiota. Because the leukemias originate in the cells of donor origin, it would be very interesting to test the donor strain that was used by Rocha's laboratory. It cannot be fully excluded that the strain used might have undergone genetic drift and lost the genetic determinants involved in leukemogenesis, as the authors themselves discuss (12). However, their mouse colonies have been "refreshed" by newly imported animals, and the original mice are no longer available (12). In this study, we addressed all sources of variability that we could assess experimentally, including genotypes, genetic backgrounds, and the possible influence of the microbiome of the mice used. Although we detected some differences, we could show that they played no role in the two relevant phenotypes for our study: thymus autonomy and leukemia. Under the new environmental conditions intrinsic to a new laboratory, it was possible not only to verify the data from the Rodewald laboratory but also to extend those findings to genotypes that had not been tested previously for thymus autonomy and/or leukemia. We covered several mouse strains that had not been covered in former studies both during the earlier stage of thymus autonomy and for T-ALL.
It has been suggested that T-ALL developing as the consequence of thymus grafting could explain the onset of leukemia in X-linked SCID (SCID-X1) patients treated by gene therapy (7). SCID-X1 patients carry loss-of-function mutations in the γc gene and require a transplant of healthy bone marrow to survive. Being a monogenetic disease that affects the hematopoietic system, SCID-X1 is a good candidate for correction by gene therapy in the absence of a compatible donor. Following this rationale, SCID-X1 has pioneered the first clinical trials of gene therapy for correction of a primary immunodeficiency (26). In total, 20 patients lacking a suitable donor enrolled in a trial for correction of the genetic defect and showed good signs of recovery and immune reconstitution, including thymus activity with de novo production of T lymphocytes for several years (27). However, 5 out of the 20 patients developed T cell leukemia as a consequence of the treatment (28, 29). This unfortunate event followed a period of years in which the patients continuously produced T lymphocytes de novo, independently of bone marrow contribution (30). We consider likely that the cellular constellation in the patients was identical to that of thymus autonomy in the thymus-transplanted mice. Nevertheless, the favored explanation for leukemia in the treated patients has been that genotoxicity due to integration of the retroviral vectors close to oncogenes led to their ectopic activation and consequent neoplastic transformation of the corrected cells (28, 29), and extensive work has followed on the improvement of better vector design (31, 32). Although the vector design is of unquestionable importance, namely for the efficiency of transduction of the target cells and safety for the patients, it is plausible that this was not the sole factor driving T-ALL in the gene therapy trials. In support of this view is the case of success for correction of adenosine deaminase–SCID (ADA-SCID) by gene therapy (33, 34). ADA-SCID is caused by deficiency in adenosine deaminase, and although the genetic and molecular defects differ from those in SCID-X1, they are both characterized by impaired lymphocyte development. Furthermore, the original vectors used in both trials were very similar (26, 35). Opposite to the treatment of SCID-X1, however, ADA-SCID patients are always myeloablated prior to the infusion of the corrected hematopoietic stem and progenitor cells, thereby effectively engrafting the corrected cells in the bone marrow. This difference suggests that the efficiency of bone marrow correction is an essential factor in the prevention of leukemogenesis emerging during treatment of SCID by gene therapy. In this line, two independent studies recently showed that the relative proportion of γc-corrected (or wild type) versus γc-deficient cells in the bone marrow is fundamental to guarantee that immune reconstitution is complete and no lymphoid malignancy emerges as a side effect (36, 37). It is becoming progressively evident that conditions that enable thymus autonomy are permissive for, and precede, leukemogenesis (38, 39). It will be important to extend these studies and understand which non–cell autonomous mechanisms regulate normal turnover and inhibit thymus autonomy, as well as understanding the cellular and molecular alterations occurring during autonomy that enable leukemogenesis. Finally, it is fundamental to limit thymus autonomy to prevent the risk of T-ALL development, and this is of particular relevance in the context of correction of immunodeficiencies.
The authors have no financial conflicts of interest.
We thank K. Xavier, J. Barata, J. Demengeot, J. Carneiro, and H.J. Fehling for critical reading of the manuscript. We acknowledge D. Zanatta and C. Alves for technical support and H.J. Fehling for the Rag2−/−γc−/− mice. We thank the Animal House Facility, the Genomics Unit, and the Histopathology Unit of the IGC for outstanding support of this work.
This work was supported by the Instituto Gulbenkian de Ciência (IGC)-Fundação Calouste Gulbenkian, by the Portuguese National Research Council (Fundação para a Ciência e Tecnologia [FCT], Grant PTDC/BIA-BID/30925/2017) (to V.C.M.). R.A.P. is a Ph.D. student of the IGC Integrative Biology and Biomedicine Ph.D. Program and is supported by an individual Ph.D. fellowship from the FCT (PD/BD/114341/2016). This work was developed with the support of the research infrastructure Congento, Project Grant LISBOA-01-0145-FEDER-022170, and ONEIDA project Grant LISBOA-01-0145-FEDER-016417, both cofinanced by Lisboa Regional Operational Programme (Lisboa 2020) under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund and FCT.
The sequences presented in this article have been submitted to the National Center for Biotechnology Information's Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/PRJNA508748) under accession number PRJNA508748.
The online version of this article contains supplemental material.
Abbreviations used in this article:
ADA-SCID
adenosine deaminase–SCID
amplicon sequence variant
C57BL/6J
γc
common cytokine receptor γ chain
double-negative
Instituto Gulbenkian de Ciência
SCID
severe combined immunodeficient
SCID-X1
X-linked SCID
single nucleotide polymorphism
T-ALL
T cell acute lymphoblastic leukemia.
Received October 10, 2018.
Accepted December 2, 2018.
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You are going to email the following T Cell Acute Lymphoblastic Leukemia as a Consequence of Thymus Autonomy
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on December 28, 2011 at 2:17 pm Ted C.
Sounds like he's a grade A D-BAG to me, Mr. Rozell. That I am not skeptical of at all. Too bad he wasn't among those within the death cars of this train. Perhaps his skepticism would deminish. But I doubt it. People like him are so full of hate, he wouldn't believe it happened if he witnessed this horror first hand. My hopes are he rots in hell.
My most sincere congratulations on your efforts to promote truth through the teaching of history, and to eradicate ignorance and the hatred and evil that spring from it.
The "SOB" who wrote this article 'did not witness' the horror that we soldiers saw at Farsleben. Very Unfortunately, George Gross did not take any close up photos of the victims who were jammed in the freight cars. The people who were in most of these photos, were from the "passenger cars" and were in fairly good condition, as compared to those who were jammed in the freight cars. The writer did not see these victims, nor did he smell the stench that emanated from these freight cars containing about 75 'skeletons" hE SHOULD HAVE BEEN THERE INSTEAD OF CRITISIZING AND SITTING IN HIS EASY CHAIR. WE WHO WERE THERE, KNOW WHAT IT WAS LIKE !!
If You were not there, DO NOT Critisize !!
Frank W. Towers, One of the initial Liberators of 'The Train' !!!


text:
Stayer Park Accessible Playground
Plymouth Lions "Dare to Dream"
In September 2016, the Plymouth Lions Club shared a vision for our Club's most ambitious community service project to date, nicknamed "Dare to Dream." As part of the City's Mullet Corridor project, the Plymouth Lions have helped to build a playground in Stayer Park that is inclusive for children of ALL ages and abilities to play, learn, and grow together. The project was completed in Fall 2017, and the official dedication took place on May 19th, 2018, at the playground and band shelter.
Stayer Park Project History
Before the Plymouth Lions Club celebrated its 50th anniversary in 2014, club members were challenged to dream of a community service project worthy of such a milestone. The project became known among club members as the "Dare to Dream" project.
The club's vision was to enhance one of the city's public parks and play areas, making it more accessible for children of all abilities. Stayer Park became the front-runner, and as we began planning, it quickly became evident that this project would require a lot more than selling grilled cheese sandwiches at the Fair to fund it.
When the City of Plymouth began considering improvements as part of the Mullet Corridor Study project in 2015, several members of the Plymouth Lions Club were invited to participate in the study and planning phases. "Dare to Dream" aligned very well with the city's vision for Stayer Park.
The Dream Took Shape
We worked with the Plymouth Parks Commission for over a year before plans were finalized. The timing also aligned with another major Lions Club milestone – the Lions Club International 100 Year Anniversary in 2017. "Dare to Dream" became our "Lions Centennial Project," with ground breaking in Spring 2017, and project completion in the Fall 2017.
Brian Yerges (left), City Administrator & Utilities Manager, and Kevin Loose (right), Plymouth Lions Club member and chairman of the "Dare to Dream" project, visited Landscape Structures in Delano, Minnesota, the company selected to design and fabricate the playground equipment for the Stayer Park expansion.


text:
Patterson's presence felt as Falcons get running game going
NFL Headlines
Titans' Henry on facing Cincy: We'll see
Eagles say Hurts is starting QB in 2022
Bengals' D-line banged up as Henry looms
Browns QB Mayfield has shoulder surgery
NFL asks Nevada to nix Gruden lawsuit
Packers surprisingly release DL Keke
Bisaccia interviews to coach '22 Raiders
Vikings interview Cowboys coordinators
Chiefs' Poles has 2nd talk with Giants
Bears interview 49ers' Carthon for GM
(AP Photo/Phelan M. Ebenhack)
By GEORGE HENRY
ATLANTA (AP) Cordarrelle Patterson made sure the Falcons finally produced a 100-yard rusher after they went 22 games without one.
It couldn't have come at a better time.
Patterson, in his ninth season and first with Atlanta, ran for a career-best 108 yards and two touchdowns on just 16 carries in Sunday's 21-14 victory at Jacksonville. It was no coincidence that they won after Patterson, the do-it-all running back-receiver-special teams standout, returned following missing one week and part of another game with an ankle injury to help the team snap a two-game skid.
The Falcons (5-6) had been outscored 68-3 in their previous two games, and they clearly weren't the same offense without Patterson. Now they're back as a long-shot hope in the NFC playoff hunt.
"I'm a big part of this offense," he said. "That's a no-brainer, man. I mean, outside looking in, you can see that. Like I told you before, I love it here. I love when I'm out there on the field. I love competing. I love having fun. That's just something I'm always doing. When I'm out there, man, I'm going to give it my all. I just love it."
Coach Arthur Smith was insistent that the Falcons, who began the game ranked 31st in rushing, could run the ball against the Jaguars. He harped on it in practice all week and told his players that it's a long season and storylines can change.
"That's what good teams have to do," Smith said. "That's why you can live in all these narratives, and they're all (phony) if you don't continue to grow and improve. We felt good about it, about the run game, regardless of what statistics and situations, whatever. We kept chipping away and today was a great job by the entire offense running the football.
"Obviously, having C.P. back adds an element. He's a warrior. He's a big, physical presence that can do a lot for us, and that's clear. It was good to have him back."
WHAT'S WORKING
The offensive line had its best showing of the season as Atlanta rushed for a season-high 149 yards. Matt Ryan was sacked just once, and that came because of tight coverage in the secondary. Smith decided to switch centers, starting Matt Hennessy as usual but subbing him for Drew Dalman as the game went on. Smith indicated Monday that the rotational plan could extend into this week.
The Falcons have no deep passing threat. Ryan almost never takes a shot downfield. Smith said the reason is the cornerbacks are playing far off, whether it's in single-high coverage or in variations of two-quarters schemes, and forcing Ryan to complete the ball underneath.
"Certainly, I think it's a trend in the NFL," Smith said. "When you see what their game plan is, we've got to adapt, and I thought we did a decent job of that yesterday."
Mykal Walker stepped in for the injured Deion Jones and had six solo stops, 11 tackles overall, one quarterback hit and one pass breakup.
Smith had no update Monday on No. 1 receiver Calvin Ridley, who missed his fifth straight game with mental health issues. Smith cut off two questions from reporters asking whether Ridley might return this season. "When we have an update, we'll give it to you," he said.
Top cornerback A.J. Terrell left the game late in the third quarter with a shoulder injury. He didn't return but said after the game he should be ready for next week. Jones was on the injury report Friday with an ailing shoulder. He had started 47 consecutive games.
3 for 3 - Atlanta scored three TDs in three trips to the red zone.
Keep beefing up the running game and get ready to face Tom Brady and the Tampa Bay Buccaneers this Sunday.
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