text:
In the first part of my somehow lengthy comparison between Fortran, ILNumerics, Matlab and numpy, I gave some categorization insight into terms related to 'performance' and 'language'. This part explains the setup and hopefully the results will fit in here as well (otherwise we'll need a third part )
This comparison is going to be easy and fair! This means, we will not attempt to compare an apple with the same apple, wrapped in a paper bag (like often done with the MKL) nor are we going to use specific features of an individual language/ framework – just to outperform another framework (like using datastructures which are better handled in a OOP language, lets say complicated graph structures or so).
We rather seek for an algorithm of:
- Sufficient size and complexity. A simple binary function like BLAS: DAXPY is not sufficient here, since it would neglect the impact of the memory management – a very important factor in .NET.
- Limited size and complexity in order to be able to implement the algorithm on all frameworks compared (in a reasonable time).
We did chose the kmeans algorithm. It only uses common array syntax, no calls to linear algebra routines (which are usually implemented in Intel's MKL among all frameworks) and – used on reasonable data sizes – comes with sufficient complexity, computational and memory demands. That way we can measure the true performance of the framework, its array implementation and the feasibility of the mathematical syntax.
Two versions of kmeans were implemented for every framework: A 'natural' version which is able to be translated into all languages with minimal differences according execution cost. A second version allows for obvious optimizations to get applied to the code according the language recommendations where applicable. All 'more clever' optimizations are left to the corresponding compiler/interpreter.
The test setup: Acer TravelMate 8472TG, Intel Core™ i5-450M processor 2.4GHz, 3MB L3 cache, 4GB DDR3 RAM, Windows 7/64Bit. All tests were targeting the x86 platform.
ILNumerics Code
The printout of the ILNumerics variant for the kmeans algorithm is shown. The code demonstrates only obligatory features for memory management: Function and loop scoping and specific typed function parameters. For clarity, the function parameter checks and loop parameter initialization parts have been abbreviated.
public static ILRetArray<double> kMeansClust (ILInArray<double> X, ILInArray<double> k, int maxIterations, bool centerInitRandom, ILOutArray<double> outCenters) { using (ILScope.Enter(X, k)) { // … (abbreviated: parameter checking, center initializiation) while (maxIterations --> 0) { for (int i = 0; i < n; i++) { using (ILScope.Enter()) { ILArray<double> minDistIdx = empty(); min(sum(abs(centers - X[full,i])), minDistIdx,1).Dispose();// ** classes[i] = minDistIdx[0]; } } for (int i = 0; i < iK; i++) { using (EnterScope()) { ILArray<double> inClass = X[full,find(classes == i)]; if (inClass.IsEmpty) { centers[full,i] = double.NaN; } else { centers[full,i] = mean(inClass,1); } } } if (allall(oldCenters == centers)) break; oldCenters.a = centers.C; } if (!object.Equals(outCenters, null)) outCenters.a = centers; return classes; } }
The algorithm iteratively assigns data points to cluster centres and recalculates the centres according to its members afterwards. The first step needs n * m * k * 3 ops, hence its effort is O(nmk). The second step only costs O(kn + mn), hence the first loop clearly dominates the algorithm. A version better taking into account available ILNumerics features, would replace the line marked with ** by the following line:
... min(distL1(centers, X[full, i]), minDistIdx, 1).Dispose(); ...
The distL1 function basically removes the need for multiple iterations over the same distance array by condensing the element subtraction, the calculation of the absolute values and its summation into one step for every centre point.
Matlab® Code
For the Matlab implementation the following code was used. Note, the existing kmeans algorithm in the stats toolbox has not been utilized, because it significantly deviates from our simple algorithm variant by more configuration options and inner functioning.
function [centers, classes] = kmeansclust (X, k, maxIterations, centerInitRandom) % .. (parameter checking and initialization abbreviated) while (maxIterations > 0) maxIterations = maxIterations - 1; for i = 1:n dist = centers - repmat(X(:,i),1,k); % *** [~, minDistIdx] = min(sum(abs(dist)),[], 2); classes(i) = minDistIdx(1); end for i = 1:k inClass = X(:,classes == i); if (isempty(inClass)) centers(:,i) = nan; else centers(:,i) = mean(inClass,2); inClassDiff = inClass - repmat(centers(:,i),1,size(inClass,2)); end end if (all(all(oldCenters == centers))) break; end oldCenters = centers; end
Again, a version better matching the performance recommendations for the language would prevent the repmat operation and reuse the single column of X for all centres in order to calculate the difference between the centres and the current data point.
... dist = bsxfun(@minus,centers,X(:,i)); ...
FORTRAN Code
In order to match our algorithm most closely, the first FORTRAN implementation simulates the optimized bsxfun
variant of Matlab and the common vector expansion in ILNumerics accordingly. The array of distances between the cluster centres and the current data point is pre-calculated for each iteration of i:
subroutine SKMEANS(X,M,N,IT,K,classes) !USE KERNEL32 !DEC$ ATTRIBUTES DLLEXPORT::SKMEANS ! DUMMIES INTEGER :: M,N,K,IT DOUBLE PRECISION, INTENT(IN) :: X(M,N) DOUBLE PRECISION, INTENT(OUT) :: classes(N) ! LOCALS DOUBLE PRECISION,ALLOCATABLE :: centers(:,:) & ,oldCenters(:,:) & ,distances(:) & ,tmpCenter(:) & ,distArr(:,:) DOUBLE PRECISION nan INTEGER S, tmpArr(1) nan = 0 nan = nan / nan ALLOCATE(centers(M,K),oldCenters(M,K),distances(K),tmpCenter(M),distArr(M,K)) centers = X(:,1:K) ! init centers: first K data points do do i = 1, N ! for every sample... do j = 1, K ! ... find its nearest cluster distArr(:,j) = X(:,i) - centers(:,j) ! ** end do distances(1:K) = sum(abs(distArr(1:M,1:K)),1) tmpArr = minloc ( distances(1:K) ) classes(i) = tmpArr(1); end do do j = 1,K ! for every cluster tmpCenter = 0; S = 0; do i = 1,N ! compute mean of all samples assigned to it if (classes(i) == j) then tmpCenter = tmpCenter + X(1:M,i); S = S + 1; end if end do if (S > 0) then centers(1:M,j) = tmpCenter / S; else centers(1:M,j) = nan; end if end do if (IT .LE. 0) then ! exit condition exit; end if IT = IT - 1; if (sum(sum(centers - oldCenters,2),1) == 0) then exit; end if oldCenters = centers; end do DEALLOCATE(centers, oldCenters,distances,tmpCenter); end subroutine SKMEANS
Another version of the first step was implemented which utilizes the memory accesses more efficiently. Its formulation relatively closely matches the 'optimized' version of ILNumerics:
... do i = 1, N ! for every sample... do j = 1, K ! ... find its nearest cluster distances(j) = sum( & abs( & X(1:M,i) - centers(1:M,j))) end do tmpArr = minloc ( distances(1:K) ) classes(i) = tmpArr(1); end do ...
numpy Code
The general variant of the kmeans algorithm in numpy is as follows:
from numpy import * def kmeans(X,k): n = size(X,1) maxit = 20 centers = X[:,0:k].copy() classes = zeros((1.,n)) oldCenters = centers.copy() for it in range(maxit): for i in range(n): dist = sum(abs(centers - X[:,i,newaxis]), axis=0) classes[0,i] = dist.argmin() for i in range(k): inClass = X[:,nonzero(classes == i)[1]] if inClass.size == 0: centers[:,i] = np.nan else: centers[:,i] = inClass.mean(axis=1) if all(oldCenters == centers): break else: oldCenters = centers.copy()
Since this framework showed the slowest execution speed of all implemented frameworks in the comparison (and due to my limited knowledge of numpys optimization recommendations) no improved version was sought.
The 7 algorithms described above were all tested against the same data set of corresponding size. Test data were evenly distributed random mumbers, generated on the fly and reused for all implementations. The problem sizes m and n and the number of clusters k are varied according the following table:
min value max value fixed parameters m 50 2000 n = 2000, k = 350 n 400 3000 m = 500, k = 350 k 10 1000 m = 500, n = 2000
By varying one value, the other variables were fixed respectively.
The results produced by all implementations were checked for identity. Each test was repeated 10 times (5 times for larger datasets) and the average of execution times were taken as test result. Minimum and maximum execution times were tracked as well.
Ok, I think we make it to the results in this part! The plots first! The runtime measures are shown in the next three figures as error bar plots:
Clearly, the numpy framework showed the worst performance – at least we did not implement any optimization for this platform. MATLAB, as expected, shows similar (long) execution times. In the case of the unoptimized algorithms, the ILNumerics implementation is able to almost catch up with the execution speed of FORTRAN. Here, the .NET implementation needs less than twice the time of the first, naïve FORTRAN algorithm. The influence of the size of n is negligible, since the most 'work' of the algorithm is done by calculating the distances of one data point to all cluster centres. Therefore, only the dimensionality of the data and the number of clusters are important here.
For the optimized versions of kmeans (the stippled lines in the figures) – especially for middle sized and larger data sets (k > 200 clusters or m > 400 dimensions) – the ILNumerics implementation runs at the same speed as the FORTRAN one. This is due to ILNumerics implementing similar optimizations into its builtin functions as the FORTRAN compiler does. Also, the efforts of circumventing around the GC by the ILNumerics memory management and preventing from bound checks in inner loops pay off here. However, there is still potential for future speed-up, since SSE extensions are (yet) not utilized.
For smaller data sets, the overhead of repeated creation of ILNumerics arrays becomes more important, which will be another target for future enhancements. Clearly visible from the plots is the high importance of the choice of algorithm. By reformulating the inner working loop, a significant improvement has been achieved for all frameworks.


text:
Just before our experts point out anything too hastily, our team need to first comprehend what I really imply by this claim. Commonly folks just grab a cam and take a breeze, see the end result and assume– hello that was effortless and also exciting. I believe I would certainly just like to do this for a lifestyle. It may sound a little fete, yet that usually takes place. Many pertain to a rude waking up relatively rapidly, however, a few of these folks succeed also. Let's look a little closer at occupation information regarding photography.
Career info concerning photography is crucial to gain access to and also concentrate on originally due to the fact that it is actually a competitive market where the job is commonly desirable to lots of folks. The professional photographer has to have "a good eye", imagination, and also some innovation to perform properly within this field. That is actually certainly not all. Photographers adore going into their personal service. Appropriately, they need to have the ability to sustain a business and to make the most of opportunities offered through swiftly transforming innovations. While finding career info regarding photography you must initially talk with one of your local area digital photographers for details. You can easily inquire concerns regarding the task market in your region and also just how much instruction was actually entailed just before he/she considered on their own ready. You'll locate your regional freelance photographer will deliver you understanding that will be valuable in many cases.
What as a freelance photographer perform you intend on achieving? A lot of would certainly acknowledge, the photographer is actually a musician, historian, as well as updates occasion individual. Possibly you had actually already intended on your career and also exactly how you want to obtain it. Occupation information regarding photography will prepare you for details projects but you should understand other components related to the job. A digital photographer produces as well as keeps photos that , narrate, or even document an activity. The successful photographer is actually called for to possess the ability to show a subject to attain a specific effect while likewise picking the suitable equipment. A great freelance photographer might enhance the subject matter's look along with all-natural or artificial illumination, shoot the subject matter from a fascinating perspective, draw attention to a particular component of the topic by tarnishing the history brendan bannister photography, or even maybe using different lens to produce preferred amounts of particular at several proximities.
This is merely an instance of some of the demands that an excellent professional photographer should possess. Job details concerning photography are going to vary according to what type of photography passions you. Undersea photography is ending up being a thrilling industry of undiscovered areas. Other digital photographers focus on places like portraiture, office and industrial, medical, headlines, or even fine arts photography.
Portrait Photographers: The one's we see usually who typically include those special family occasions. They commonly take individual photos, team photos and often do work in their very own workshops. A ton of photographers specialize in taking wedding ceremony pictures, spiritual events, or university pictures.
Commercial and also Industrial: Career information regarding photography includes professionals that specialize in business and also industrial photography. These photographers take images of different subjects, such as buildings, models, stock, artifacts, as well as gardens. Why would certainly a digital photographer go around taking these images? This is actually a customized area wherein a ton of money may be created. The pictures are actually used in a range of media, including books, files, advertisements, and catalogs. The industrial professional photographer is generally hired earlier as well as recognizes what his assignment is going to be. He performs certainly not take photos and also try to find a shopper. The industrial professional photographer takes pictures of machinery, products, workers, and also business officials. Accounts taken are actually often for a certain reason. In the future these pictures help developer's review tasks, promotion, or files of devices development or release, such as positioning of an overseas oil well. Area of Science: Career info regarding photography prevails in the field of science. The audio of medical information and also treatments is actually typically carried out by a freelance photographer. The industry of uncommon phenomena or sometimes called ghost seeking utilizes unique sort of equipment and is actually important to aid confirm the life of perhaps a haunting. A lot of things grow when you function in the medical industry that is why a professional photographer must have expertise in one or more areas of design, medication, biology, or chemical make up.

subset: commoncrawl


text:
MP3 download Teen boy puts religion in his genes by Ian Woolf, Twenty years of Diffusion, a walk down memory lane by Ian Woolf Fact and sound checking by Charles Willock, Produced and hosted by Ian Woolf. The first injection...
Posted by Ian Woolf on December 17, 2018 9:28 PM
If you use an RSS reader, you can subscribe to a feed of all future entries tagged 'Bible'. [What is this?]
Other tags used on this blog:


text:
Last week Forrester released a report advising most marketers wait to use location-based social networks (LBSN) as only 4% of the US population is currently using platforms such as Foursquare (the current market leader), and that the networks skew heavily male. They advise that brands that target young males experiment with the services and other brands adopt a "wait and see" approach.
I couldn't disagree more. Here are my 5 reasons why it's smart to start experimenting now.
1. First Movers.
There's something to be said for getting a head start on your competition in the digital space. Brands like Starbucks, Dell, Pepsi, and Nike have all taken advantage of the emerging channels and reaped the rewards of building a strong early foundation with consumers.
While you should not rush into a new tool without understanding your strategic goals and how it integrates with your business objectives, experimenting with emerging technologies that are opt-in and potentially have a direct customer impact is smart.
When Facebook opened their gates to the general population in 2006 they had a small user base of university students. Four years later they are a behemoth. Twitter adoption rates have been increasing exponentially year over year since their launch in 2007 and the tool is now considered a "must use" for social business. Considering Foursquare launched about a year ago, can we expect to see the same type of growth curve as the early adopters begin to influence the early majority? (see "Crossing the Chasm" adoption curve)
2. Google. Facebook. Oh My.
Location-based services are not limited to the current apps we have been hearing about. Facebook has expressed they will add a location-based offering soon, Twitter has added "Tweet with your location" to their service, and the biggest news is that Google is adding a Places API to their eco-system, as well as adding LB data extensions to their mobile advertising product.
LBSN will become mainstream sooner rather than later, and it will be the big players, not the niche networks that will drive the adoption. Testing and learning now, before it becomes ubiquitous should be something on every marketers radar.
3. Data and utility.
There is an enormous amount of insightful and actionable data that can be gleaned about your customers and prospects from mobile & LBSNs. Eventually this data could be used to inform inventory control, staffing levels, consumer tastes and trends, etc. The data can also be used in loyalty programs, to identify influencers, test new products, and as real-time service focus groups.
Companies already testing the waters include:
Nike with True City; Starbucks with their Foursquare offers; The Pepsi mobile branded app; and the City of Chicago with their Tourism campaign.
4. Sales, Coupons, Offers, and more.
Part of the Forrester analysis identified that mobile couponing is widely successful with the users currently using the services, which is interesting as the base is primarily young males, not the average coupon-consuming demographic. Gone are the days of clipping coupons in the Sunday paper, now you can serve relevant offers and drive foot traffic and purchase directly to a mobile device. These offers are opt-in, and contextually relevant, not SMS spam. Testing offers, tips, and messaging via mobile should be on every retailers plan for the next year.
Of course one size doesn't fit all and ensuring that your product or service fits within the make-up of the demographic, depending on service (existing or branded), is a must.
5. Mobile usage.
Of course mobile, and specifically smartphone, usage is soaring year over year. Ignoring mobile at this point is like ignoring the Internet in 2002 because broadband wasn't prevalent yet.
Bottom line for marketers:
Experiment. See what fits, what your customers are looking for, and where you can add value. Don't wait until it becomes mainstream, because that will be sooner than you think and you'll be playing catch-up.
[cross-posted from Teehan+Lax]


text:
Updated July 29, 2021 at 6:00 PM ET
Scarlett Johansson is suing the Walt Disney Co. for releasing her movie Black Widow on streaming and in theaters at the same time.
Black Widow opened in theaters two weeks ago to the biggest box office numbers of the coronavirus pandemic: $80 million on its opening weekend.
After the first weekend, the numbers fell off a cliff. Industry observers suggest that one reason attendance fell so quickly is that Disney had screened the film simultaneously on Disney+, allowing audiences to bypass theaters and watch it at home.
Johannsson is the film's star and also its executive producer. She says in her lawsuit — which was first reported by The Wall Street Journal — that her agreement with Disney's Marvel Entertainment guaranteed an exclusive theatrical release and based her salary in part on the film's box office performance.
Disney responds that the lawsuit has no merit
A Disney spokesperson responded, according to Entertainment Weekly, by saying that Disney "has fully complied with Ms. Johansson's contract and furthermore, the release of Black Widow on Disney+ with Premier Access has significantly enhanced her ability to earn additional compensation on top of the $20M she has received to date."
With the pandemic scrambling Hollywood's release patterns, the exclusive theatrical opening — long the industry norm — has lately been threatened.
A few big films — F9, for instance, and the upcoming 007 movie No Time to Die— delayed their premieres so they could go the traditional route. Others, like Pixar's Soul, bypassed theaters entirely and went straight to streaming.
WarnerMedia, to boost its streaming service HBO Max, is releasing all of its titles this year simultaneously in cinemas and on HBO Max. But it renegotiated many of its talent contracts to do so, reportedly paying stars and directors more than $200 million.
Johansson's complaint says her representatives tried to renegotiate her contract but Disney and Marvel were unresponsive.
Copyright 2023 NPR. To see more, visit https://www.npr.org.


text:
Chronic Pain Relief
When an injury occurs, pain signals are sent from the injured area along the spinal cord and then to the brain. As the injury begins to heal, pain usually becomes less severe. However, chronic pain is different. The body continues to send pain signals to the brain, even if the injury has healed. Chronic pain can last several weeks to several years. It limits mobility and makes it very challenging to get through daily tasks and participate in normal activities. Chronic pain may feel dull or sharp and be steady or intermittent. In addition, it can occur in any part of the body.
Common types of chronic pain include lower back pain, arthritis pain, and neurogenic pain. Usually, chronic pain is caused by an injury, such as a pulled muscle or back sprain. Many medical professionals feel that chronic pain develops from damaged nerves. Chronic pain affects more than 1 billion people in the world. It's quite a global problem.
Physical Therapy and Chronic Pain
Since chronic pain comes in many forms, it's important that a treatment plan is personalized. Physical therapy is personalized. A physical therapist will do a comprehensive exam to assess the root cause of chronic pain. Using that information, a physical therapist will customize an individualized therapy plan. Physical therapy services are comprised of passive and active treatments. Active treatments are things like therapeutic exercises. Passive treatments are ones where the patient does not actively participate.
Examples of passive treatments include deep tissue massage, hot and cold treatments, transcutaneous electrical nerve stimulation (TENS), and ultrasound. With deep tissue massage, a physical therapist uses direct pressure to release tension in muscles, tendons, and ligaments. Physical therapists also alternate between hot and cold therapies. With heat therapy, more blood and oxygen are brought to the affected area. With cold therapy, inflammation and muscle spasms are reduced. TENS stimulates the muscles and increases endorphins to reduce pain. Ultrasound increases blood circulation and reduces chronic pain. Passive therapies are usually done hand-in-hand with active therapies.
In addition, physical therapists teach patients how to incorporate ergonomic principles into daily life. They may also recommend a personalized home exercise program as part of pain management. The goal of physical therapy is to reduce pain and to increase strength flexibility gradually. It is also intended to manage pain, sustain long-term recovery, and prevent the future recurrence of pain.
A physical therapist works closely with the patient in goal setting. Patients and physical therapists work out a goal-setting chart for activities, record progress, and note any difficulties. This way, progress can be monitored, and changes can be made when necessary. Physical therapists incorporate behavioral and cognitive principles into rehabilitation for chronic pain. It's a collaborative approach to chronic pain. The process of overcoming chronic pain can be long and complex. Rehabilitation involves overcoming physical obstacles, and physical therapists play an important role in chronic pain management. They help patients address obstacles with chronic pain and provide support. Free Consultation at Toledo, Sylvania, Perrysburg, Maumee & Swanton, OH centers, or call us at 419-559-5591.


text:
- Research article
- Open access
- Published:
Aberrant CD200/CD200R1 expression and function in systemic lupus erythematosus contributes to abnormal T-cell responsiveness and dendritic cell activity
Arthritis Research & Therapy volume 14, Article number: R123 (2012)
CD200 is a type I transmembrane glycoprotein that can regulate the activation threshold of inflammatory immune responses, polarize cytokine production, and maintain immune homeostasis. We therefore evaluated the functional status of CD200/CD200 receptor 1 (CD200R1) interactions in subjects with systemic lupus erythematosus (SLE).
Serum CD200 level was detected by ELISA. The expression of CD200/CD200R1 by CD4+ T cells and dendritic cells (DCs) was examined by flow cytometry, and then compared between SLE patients and healthy controls. Peripheral blood mononuclear cells were stained with carboxyfluorescein diacetate succinimidyl ester and annexin V/propidium iodide for evaluation of the effect of CD200 on cell proliferation and apoptosis. In addition, the effect of CD200 on DC function was determined by transwell migration assay as well as by measurement of binding and phagocytosis of apoptotic cells.
In SLE patients, the number of CD200+ cells and the level of soluble CD200 were significantly higher than in healthy controls, whereas the expression of CD200R1 by CD4+ T cells and DCs was decreased. Furthermore, the increased CD200 expression by early apoptotic cells contributed to their diminished binding and phagocytosis by DCs in SLE. Importantly, the engagement of CD200 receptor on CD4+ T cells with CD200-Fc fusion protein in vitro reduced the differentiation of T-helper type 17 cells and reversed the defective induction of CD4+CD25highFoxP3+ T cells by transforming growth factor beta in SLE patients. Conversely, blockade of CD200-CD200R1 interaction with anti-CD200R1 antibody promoted CD4+ T-cell proliferation.
CD200 and CD200R1 expression and function are abnormal in SLE and may contribute to the immunologic abnormalities in SLE.
Systemic lupus erythematosus (SLE) is an autoimmune disease that affects many tissues and organs. The major immunopathological findings of SLE include defective immune regulation with the breakdown of immune tolerance, autoantibody formation followed by immune complex deposition, cytokine imbalance, and inflammation [1]. Failure of phagocytes to remove apoptotic cells has been suggested to allow excessive release of autoantigens and to lead to the induction of autoimmunity, although the underlying mechanisms remain unclear [2–6]. In addition, CD4+CD25highFoxP3+ regulatory T cells (Tregs), which are pivotal in the maintenance of T-cell homeostasis and are critical regulators of immune tolerance [7, 8], exhibit quantitative and/or qualitative deficiencies in SLE that may contribute to the development of lupus pathogenesis [9, 10].
CD200 is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily, and is expressed by a variety of cells, including B cells, activated T cells, follicular dendritic cells (DCs), and neurons [11–13]. CD200 consists of extracellular, transmembrane, and intracellular domains, although its intracellular region lacks a signaling motif [11–13]. CD200 receptors include CD200R1 to CD200R4, of which CD200R1 has the highest binding affinity [14]. The distribution of CD200 receptors is mainly on myeloid-derived cells, such as DCs, macrophages, and also activated T cells [15, 16]. The known immunoregulatory roles of the CD200/CD200R1 pathway include suppression of the degranulation of mast cells and basophils [17] and negative regulation of macrophage function [18].
Hoek and colleagues found that CD200-deficient mice had increased endogenous activation of macrophages/myeloid cells in the central nervous system, with enhanced susceptibility to experimental allergic encephalomyelitis and collagen-induced arthritis [18]. Administration of CD200R-Ig to disrupt CD200-CD200R interaction also increased the susceptibility of mice to collagen-induced arthritis. Furthermore, Broderick and colleagues reported that blockade of CD200 resulted in the early onset of experimental autoimmune uveoretinitis in mice [19]. In addition, Rosenblum and colleagues studied CD200-knockout mice in a model of UV-mediated induction of tolerance to hapten, and suggested that the expression of CD200 in skin cells plays a role in autoimmune congenital alopecia [20]. Finally, Gorczynski and colleagues showed that tumor growth in vivo can be monitored by levels of soluble CD200 in serum of tumor-bearing animals [21], whereas Moreaux and colleagues found significant overexpression of CD200 in a variety of cancers compared with normal cells or tissues and suggested that CD200 might be a potential therapeutic target and prognostic factor for a large array of malignancies [22].
While available evidence highlighted an important role of CD200/CD200R1 in experimental autoimmune diseases, the role of CD200/CD200R1 in human autoimmune diseases such as SLE remains unknown. We therefore explored the expression and function of CD200/CD200R1 in subjects with SLE.
Materials and methods
Patients and healthy controls
Altogether, a total of 161 new-onset untreated patients fulfilling the American College of Rheumatology classification criteria for SLE were enrolled in this study. All were female, and their age ranged from 12 to 55 years with a mean age of 29.0 ± 10.2 years (see Additional file 1). Ninety-five gender-matched and age-matched healthy volunteers were recruited as healthy controls (HCs). The Ethics Committee of Peking Union Medical College Hospital approved this study and informed consent was obtained from each patient and HC.
Antibodies and reagents
mAbs targeting the following molecules were used, either unlabeled or as fluorescein isothiocyanate, phycoerythrin, allophycocyanin, or phycoerythrin-cyanin 7 conjugates: CD4 (RPA-T4), CD11c (3.9), CD25 (BC96), CD200R (OX-108), CD38 (HIT2), and CD123 (6H6) from BioLegend (San Diego, CA, USA); IFNγ (B27), CD3 (UCHT1), and CD28 (CD28.2) from BD Pharmingen (Franklin Lakes, NJ, USA); and CD200 (OX104), IL-4 (MP4-25), IL-17 (eBio64DEC17), and Foxp3 (PCH101) from eBioscience (San Diego, CA, USA). In all experiments, control mAbs of the respective IgG were included.
The CD200 Duoset and B-cell activating factor belonging to the TNF family, IFNα, and IL-6 ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). The Cell Trace™ carboxyfluorescein diacetate succinimidyl ester cell proliferation kit was obtained from Invitrogen (Carlsbad, CA, USA). Recombinant IL-2, IL-4, transforming growth factor beta (TGF-β) 1, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were purchased from PeproTech Inc. (Rocky Hill, NJ, USA); PKH26, PKH67, ionomycin, and phorbol-12-myristate-13-acetate were obtained from Sigma-Aldrich (St Louis, MO, USA); the Vybrant Apoptosis Assay Kit #2 was obtained from Invitrogen; and recombinant human CD200-Fc, anti-human CD200 R1 antibody, and human IgG control were purchased from R&D Systems.
Enzyme-linked immunosorbent assay
The levels of serum CD200, IL-6, IFNα, and B-cell activating factor belonging to the TNF family were detected with ELISA kits according to the manufacturer's instructions.
Real-time polymerase chain reaction
Total RNA was isolated from peripheral blood mononuclear cells (PBMC) of SLE patients and HCs with TRIzol (Invitrogen), first-strand cDNA was synthesized, and the DNA amplification was detected with the dye SYBR Green (TAKARA, Otsu, Japan). Primers were as follows: for CD200, 5'-CCGTCAACAAAGGCTATTGG-3' (forward) and 5'-ATTTAGGGCTCTCGGTCCTG-3' (reverse); for CD200R1, 5'-GACCAGAGAGGGTCTCACCA-3' (forward) and 5'-TTGAAGCGGCCACTAAGAAG-3' (reverse); and for glyceraldehyde-3-phosphate dehydrogenase, 5'-ACTTCAACAGCGACACCCACT-3' (forward) and 5'-GCCAAATTCGTTGTCATACCAG-3' (reverse).
Cell culture, stimulation and treatment
Cells were cultured in RPMI 1640 medium supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin, 0.05 mM nonessential amino acids, 2 mM L-glutamine, as well as 10% heat-inactivated FCS in a humidified carbon dioxide-containing atmosphere at 37°C. Cells were stimulated with anti-CD3 and anti-CD28 mAbs at 1 μg/ml, respectively. Recombinant human CD200-Fc, anti-human CD200R1 antibody, and human IgG control were used at 100 ng/ml; and recombinant human IL-2, TGF-β, IL-4, and GM-CSF were used at 20 ng/ml, 2 ng/ml, 100 ng/ml, and 100 ng/ml, respectively. For T-cell differentiation experiments, PBMC were co-cultured with CD200-Fc or anti-CD200R1 for 48 hours. Golgistop was added in the presence of phorbol-12-myristate-13-acetate and ionomycin 5 hours before cells were collected and stained for membrane molecules. Intracellular staining for IL-17, IL-4, and IFNγ was also performed after fixation and permeabilization with fixation/permeabilization buffer. For cell proliferation assays, PBMC were stained with 5 μM carboxyfluorescein diacetate succinimidyl ester, stimulated by anti-CD3 and anti-CD28 mAbs alone or in the presence of CD200-Fc or anti-CD200R1, and cell proliferation was measured on day 5 by flow cytometry. The cell division index - which was defined as the ratio of the proportion of proliferated cells with decreased carboxyfluorescein diacetate succinimidyl ester fluorescence after stimulation to that without stimulation - was analyzed.
Cell separation
CD4+CD25- T cells and CD14+ monocytes were isolated using a CD4+CD25+ Regulatory T Cell Isolation Kit (130-091-301; Miltenyi Biotec) and CD14 MicroBeads (130-050-201; Miltenyi Biotec) according to the manufacturer's instructions. PBMC were induced to undergo apoptosis by X-ray accelerator (5 Gray) [23] and cultured in complete RPMI 1640 medium in a humidified carbon dioxide-containing atmosphere at 37°C for 48 hours. Apoptosis and necrosis were detected by annexin V and propidium iodide (PI) staining according to the manufacturer's protocol. CD200+ live cells, CD200- live cells, CD200+ apoptotic cells, CD200- apoptotic cells, and necrotic cells were sorted by flow cytometry using the Moflo (Cytomation, Fort Collins, Colorado, USA). The sort gates were additionally restricted to the lymphocyte gate as determined by typical forward and sideward scatter characteristics [24, 25].
Generation of dendritic cells
Monocytes were cultured with recombinant human GM-CSF (100 ng/ml) and recombinant human IL-4 (100 ng/ml) and were harvested after 6 days. The monocyte-derived DCs were used for co-culture experiments and transwell assays.
Cell staining and co-culture
The monocyte-derived DCs and apoptotic and necrotic cell targets were labeled with green fluorescent dye PKH67 and red fluorescent dye PKH26 (Sigma-Aldrich), respectively, and then co-cultured (cell ratio 104:104) for 3 hours, after which they were analyzed by fluorescence microscopy and flow cytometry.
Transwell migration assay
The monocyte-derived DCs were seeded in the upper chambers of the transwell. The lower chambers were filled with IgG control (100 ng/ml), CD200Fc (100 ng/ml), RANTES (50 ng/ml) or CD200Fc plus RANTES. After 8 hours of incubation, the cells that had migrated to the lower chamber were counted.
Western blot
CD4+ T cells were cultured with recombinant human CD200Fc (IgG as control). After 5 days, cells were harvested, washed twice in ice-cold PBS, and lysed by incubation for 1 hour in a buffer containing 20 mM HEPES (pH 7.9), 20% glycerol, 1% Nonidet P-40, 1 mM MgCl2, 0.5 mM ethylenediamine tetraacetic acid, 0.1 mM ethyleneglycol tetraacetic acid, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and a proteinase inhibitor cocktail (BD Biosciences, Franklin Lakes, NJ, USA). Lysates were kept on ice and vortexed every 10 minutes for 1 hour before centrifugation at 13,000 rpm and 4°C. Equal amounts of protein were separated by SDS-PAGE (Invitrogen) and transferred to Immobilon PVDF membranes (Millipore, Billerica, Massachusetts, USA) and were blocked with 5% dry milk in PBS containing 0.5% Tween 20 before incubation with specific antibodies against downstream of tyrosine kinase 2 (DOK2; ab47507) or phosphorylated DOK2 (phospho Y299) (ab47376; Abcam, Cambridge, UK), followed by incubation with horseradish peroxidase-conjugated secondary antibody and development using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Inc. Waltham, Massachusetts, USA).
Statistical analysis
All data were analyzed using SPSS 13.0 software. Data that passed both the Kolmogorov-Smirnov and the Shapira-Wilk tests (P > 0.05) were considered in a normal distribution. For data with normal distribution and homogeneity of variance (means and standard deviations), one-way analysis of variance with adjusted Bonferroni correction was used to assess the differences among groups. An independent-sample t test and a paired-sample t test were used to compare differences between two groups and differences before and after treatment. Correlation was calculated with Pearson's correlation. For data with a non-normal distribution (medians and 25th to 75th percentiles), the Mann-Whitney test was used to compare differences between two groups and correlation was analyzed with Spearman's rank-order test. P < 0.05 was considered statistically significant.
Increased CD200 but decreased CD200R1 expression by CD4+T cells and dendritic cells in SLE patients
CD200 and CD200R1 expression was analyzed in SLE patients and HCs. The proportion of CD200+ cells in PBMC of SLE patients was significantly higher than that in HCs (Figure 1A, B), especially in the CD4+ T-cell population, the CD11c-CD123high plasmacytoid DCs, and the CD11c+CD123- myeloid DCs (Figure 1B; see Additional file 2), but not in CD8+ T cells, CD19+ B cells, CD38bright plasma cells (data not shown), or CD14+ monocytes (P > 0.05). The percentage of CD3+CD200+ cells was negatively correlated with serum complement 3 (r = -0.486, P < 0.05; Figure 2A). Unlike the cell numbers, there was no significant difference in the mean fluorescence intensities of CD200 expression between SLE patients and HCs in the different cell subgroups (P > 0.05). Finally, even though the frequency of CD200-expressing cells was increased in SLE, CD200 mRNA expression in PBMC was significantly lower in SLE patients than in HCs (Figure 1E).
In keeping with the increased percentage of cells expressing CD200, the circulating levels of CD200 in SLE patients were also significantly higher than that in HCs (median 142.22, interquartile range 82.26 to 469.29 vs. 76.62, 45.9 to 124.93 pg/ml; P = 0.001). Furthermore, the serum CD200 level negatively correlated with the serum complement 3 level (r = -0.45, P = 0.014; Figure 2B), but not the Systemic Lupus Erythematosus Disease Activity Index score or the levels of serum B-cell activating factor belonging to the TNF family, IL-6, IFNα, or anti-dsDNA (P > 0.05; see Additional file 3).
In contrast to CD200 expression, SLE patients had a decreased proportion of CD200R1+ cells in PBMC compared with HCs. This reduction was evident in CD4+ T cells, plasmacytoid DCs, and myeloid DCs (Figure 1C, D). CD200R1 mRNA expression in PBMC was also significantly lower in SLE patients than in HCs (Figure 1E). Of note, CD4+CD45RA+ naïve T cells had less CD200R1 expression than CD4+CD45RO+ memory T cells in both HCs and SLE patients (P < 0.05), and there was no significant difference between SLE patients and HCs (see Additional file 4).
Anti-CD200R1 antibody promotes T-cell receptor-driven proliferation of CD4+T cells in SLE patients
Next we set out to determine whether the defective CD200R1 expression by CD4+ T cells affects CD4+ T-cell function in SLE patients. As engagement of CD200R1 by CD200 is known to initiate signaling by inducing phosphorylation of docking protein 2 or the adaptor protein DOK2 (p56doc-2) [26], we initially determined whether soluble CD200Fc could influence CD200/CD200R1 signaling in CD4+ T cells by examining its effect on phosphorylation of DOK2. We found that CD200Fc induced phosphorylation of DOK2 in CD4+ T cells. This result is consistent with previously reported findings and indicates that soluble CD200Fc by engaging CD200R is an agonist of the CD200/CD200R1 signaling pathway, whereas anti-CD200R1 antibody, according to the product instructions, is an antagonist and can block the receptor-ligand interaction (see Additional file 5).
Understanding the action of these reagents, we next examined their effects on the proliferation of CD4+ T cells. We found after stimulation with anti-CD3/CD28 that CD200-Fc did not cause a significant change in cellular proliferation (evaluated by the cell division index) in either SLE patients or HCs (P > 0.05; Figure 3A). However, anti-CD200R1 promoted anti-CD3/CD28-stimulated proliferation of SLE CD4+ T cells in a concentration-dependent manner. In contrast, no effect was observed in HCs (Figure 3B). The cell division indexes in SLE T cells stimulated with anti-CD3/CD28 plus control IgG and with anti-CD3/CD28 plus anti-CD200R1 antibody at 20 ng/ml or 100 ng/ml were 0.87 ± 0.54, 1.43 ± 0.92, and 2.34 ± 1.85, respectively (P < 0.05). From this result, we concluded that antagonistic anti-CD200R1 antibody promoted T-cell receptor-driven proliferation of CD4+ T cells in SLE patients, implying that endogenous CD200-CD200R1 interactions limited T-cell proliferation in SLE patients.
CD200 reduces CD4+T-cell differentiation into T-helper type 17 cells
The CD200/CD200R1 pathway has been suggested to affect the balance of cytokines, by repressing IL-2 (IFNγ) production and promoting IL-4 (IL-10) production, and to participate in transplantation tolerance [27]. We therefore investigated whether CD200-CD200R1 signaling could affect human CD4+ T-cell differentiation. We found that, while the effect of the CD200-CD200R1 pathway on the T cells in HCs was negligible (see Additional file 6), CD200-Fc but not anti-CD200R1 reduced the percentage of Th17 cells (P < 0.05; Figure 4) in SLE patients, suggesting a role for CD200/CD200R signaling in regulating Th17 cell differentiation.
CD200 signaling rescues the defective generation of CD4+CD25highFoxP3+T cells in SLE patients
In a mouse collagen-induced arthritis model, the interaction between CD200 and CD200R1 resulted in direct suppression of autoreactivity, and fostered the development of Foxp3+ regulatory T cells [28]. TGF-β induces Foxp3 gene expression in T-cell receptor-stimulated CD4+CD25- naïve T cells, which mediates their transition toward a regulatory T-cell phenotype with potent immunosuppressive potential [29]. We therefore examined whether CD200 played a role in the induction of Tregs in SLE patients. CD4+CD25- T cells were sorted from PBMC of SLE or HCs and were cultured with anti-CD3/CD28, IL-2 and TGF-β in the absence or presence of CD200-Fc or anti-CD200R1 for 7 days. We found that TGF-β induced CD4+CD25highFoxP3+ T cells from peripheral CD4+CD25- T cells in HCs, but not in SLE patients. Notably, CD200Fc but not anti-CD200R1 rescued the defective induction of CD4+CD25highFoxP3+ T cells in SLE patients (Figure 5). In SLE patients, the percentages of induced CD4+CD25highFoxp3+ T cells in TGF-β + IgG and TGF-β + CD200Fc groups were 0.93 ± 0.50% and 6.23 ± 0.72% (P = 0.013). We therefore concluded that the defective generation of CD4+CD25highFoxP3+ T cells in SLE patients could be rescued by enhanced CD200 signaling. This result suggests that the decreased expression of CD200R1 in SLE may contribute to the defective generation of Tregs.
Increased lymphocyte apoptosis with upregulation of CD200 expression in SLE
Next we were interested in determining whether CD200-CD200R interactions might also affect the activity of dendritic cells in SLE, and specifically their capacity to interact with apoptotic cells. Increased lymphocyte apoptosis and defective phagocytic removal of apoptotic cells have been suggested to contribute to the development of SLE [30–32]. We therefore initially examined whether CD200 expression by apoptotic cells was abnormal in SLE. Consistent with previous studies [4, 5, 33], we demonstrated that the percentage of spontaneous early apoptotic lymphocytes (denoted as annexin V+PI-) in PBMC from SLE patients was significantly higher than that in HCs (Figure 6A). Interestingly, we found that CD200 expression by early apoptotic cells was significantly higher compared with that expressed by live cells (annexin V-PI-), especially in SLE patients. By comparison, there was no increase in CD4 expression on apoptotic T cells detected with a mAb labeled with the same fluorochrome used to detect CD200 (Figure 7). The CD200 relative expression ratio, defined as the ratio of CD200 positivity on early apoptotic cells compared with live cells, was significantly increased in SLE patients compared with HCs (Figure 6B).
We next investigated whether CD200 expression on apoptotic cells affected their binding and phagocytosis by DCs. We obtained immature DCs by culturing human monocytes with IL-4 and GM-CSF for 6 days [34]. The immature DCs (PKH67-labeled) were then co-cultured for 3 hours with different target cell populations (PKH26-labeled), including CD200+/CD200- apoptotic cells induced by irradiation and CD200+/CD200- live cells, and were examined for cellular binding and uptake. We found that the proportion of DCs which bound and ingested apoptotic cells was higher compared with live cells. Importantly, CD200 expression on the target cells was associated with reduced binding and phagocytosis of apoptotic cells by DCs (Figures 8 and 9A). The percentages of DCs that bound and ingested CD200- versus CD200+ apoptotic cells were 44.54 ± 4.33% versus 36.76 ± 6.09% by fluorescence microscopy (P = 0.037). By flow cytometry, the percentages of DCs that ingested CD200- versus CD200+ apoptotic cells - demonstrated as PKH26 and PKH67 double-positive events - were 31.60 ± 22.98% versus 21.71 ± 20.20% (P = 0.046). The results suggested that CD200 expression on early apoptotic cells is associated with decreased binding and phagocytosis by DCs.
CD200Fc inhibits dendritic cell migration
We were interested in whether CD200-CD200R interactions might affect other functional activities of DCs, and therefore examined the impact on DC migration. In initial experiments, we noted a potential effect of soluble CD200Fc itself in reducing spontaneous DC migration - although the effect was not statistically significant (IgG vs. CD200Fc, 6.21 ± 2.23% vs. 4.38 ± 2.42% migrated; P = 0.224). However, CD200Fc significantly blocked RANTES-induced DC migration (IgG vs. CD200Fc, 10.32 ± 2.94% vs. 7.16 ± 2.36% migrated; P = 0.029; Figure 9B).
Despite the data in animal models - including collagen-induced arthritis and experimental allergic encephalomyelitis - suggesting that CD200/CD200R1 may play a role in prevention of autoimmune diseases, information on the role of the CD200/CD200R axis in human diseases - especially in SLE - is very limited. Our study demonstrated that the percentage of CD200+ cells in CD4+ T cells, plasmacytoid DCs and myeloid DCs of SLE patients was significantly higher than that for HCs. In addition, serum levels of CD200 in SLE patients were also significantly higher than those for HCs.
As CD200 lacks an intracellular signaling motif, most - if not all - of its immunological function relates to its capacity to engage and signal via its receptors, of which CD200R1 seems to be most prominent. Functional studies confirmed this by showing that CD200Fc induced phosphorylation of DOK2 in CD4+ T cells. Notably, we found that CD200R1 expression in SLE patients was significantly lower than HCs in CD4+ T cells and DCs. The dysregulated expression of CD200/CD200R1 in SLE had functional consequences since CD4+ T-cell proliferation was increased by blocking CD200R1 with specific antibody, whereas DC migration and Th17 cell differentiation were decreased and Treg generation was enhanced by engaging CD200R with CD200Fc. These results are all consistent with the conclusion that the deranged expression of both CD200 and CD200R1 in SLE contributes to the functional abnormalities characteristic of this autoimmune disease. Notably, most of the activity of CD200-CD200R1 engagement is usually believed to relate to inhibiting the activity of myeloid cell function [35]. However, we found that CD200R1 expression was also decreased on CD4+ T cells and at least the activity in regulating Tregs appeared to involve a direct effect on T cells. These findings suggest a broader spectrum of activity of CD200R1 signaling than has previously been appreciated.
Overproduction of autoantibodies in SLE is believed to be caused by insufficient removal of apoptotic cells and material by macrophages and DCs. Our study demonstrated that SLE patients had a higher proportion of spontaneous early apoptotic lymphocytes compared with HCs. The amount of apoptotic material in SLE patients may exceed the capacity of macrophages to remove it, allowing DCs to become involved in the process of apoptotic cell clearance. Under these circumstances, DCs can become either tolerogenic or stimulatory, depending on the nature of the receptors employed and the available cytokines. As CD200 expression on early apoptotic lymphocytes was increased in SLE patients, we examined whether the increased expression of CD200 on early apoptotic lymphocytes might have had an effect on their binding and uptake by DCs. We demonstrated that early apoptotic cells were more likely to be bound and engulfed by DCs than living cells. The explanation for this could be that although early apoptotic cells remain morphologically intact, specific signals - such as expression of lysophosphatidylcholine - were upregulated on the cell surface, which mediated recognition by DCs and macrophages [36, 37]. Our study also revealed that the binding and phagocytosis of early apoptotic cells that were CD200-positive were lower than those that did not express CD200, suggesting that CD200 expression in SLE could provide a signal to DCs - presumably by binding CD200R, which limits their capacity to bind and ingest apoptotic material. Aberrant expression of CD200 may therefore contribute to the decreased clearance of apoptotic material found in SLE.
To function, CD200 needs to bind to CD200R on cell surfaces. Our data confirmed that T cells expressed CD200R1. Since a previous animal study suggested that CD200/CD200R signaling had an effect on cytokine balance [27], we investigated whether CD200/CD200R1 could affect the balance of T-cell subsets in SLE. We found that CD200Fc reduced Th17 cell differentiation in SLE, but not in HCs. These results suggest that Th17 cell differentiation in SLE may be regulated by engagement of CD200R, such that signaling through this pathway limited Th17 cell differentiation. It is possible that the downregulation of CD200R in SLE resulted in less regulation of Th17 cell differentiation, which could be corrected by the increased availability of CD200. On the contrary, anti-CD3/CD28-stimulated T-cell proliferation was promoted by antagonistic anti-CD200R1 in a concentration-dependent manner in SLE patients but not HCs, suggesting that anti-CD200R1 may block the endogenous signal provided by increased expression of CD200 and, thereby, permit increased CD4+ T-cell proliferation. In summary, these results indicate that the CD200-CD200R1 pathway exerts a number of regulatory influences on T-cell function, either directly or through the action of DCs, and that the dysregulation of surface expression of these molecules may contribute to some of the immunoregulatory abnormalities characteristic of SLE.
In untreated active SLE patients, the proportion of CD4+CD25highFoxP3+ Tregs was significantly lower than in HCs (see Additional file 7). Pallasch and colleagues demonstrated that antagonistic anti-CD200 antibody could promote chronic lymphocytic leukemia cell-induced proliferation of antigen-specific T cells and reduce the proportion of CD4+CD25highFoxP3+ cells [38]. Gorczynski and colleagues showed that, in BL/6 bone-marrow cells, anti-CD200R2/3 mAb (not CD200R1) could promote the development of tolerogenic DCs through a TGF-β-dependent (but not IL-10) and CTLA-4-dependent pathway, induce more CD4+CD25highFoxP3+ Tregs, and inhibit the mixed lymphocyte reaction in a MHC-restricted and antigen-specific manner [39]. These results all suggested that the activation of the CD200/CD200R axis could exert an immunosuppressive function via promoting Treg proliferation and inhibiting effector T-cell function. Our study found that TGF-β induced generation of CD4+CD25highFoxP3+ T cells in HCs, whereas this was not seen in SLE patients. This finding is consistent with a previous study that demonstrated defective expression of TGF-β signal transduction molecules in most SLE patients [40]. Interestingly, we found that the addition of CD200Fc rescued the defective generation of CD4+CD25highFoxP3+ T cells in SLE patients, indicating that CD200 could intervene in the TGF-β signaling pathway and promote Treg generation. This effect appeared to be directly mediated by T cell-T cell interaction since these studies were carried out with sorted T cells. Specific signals and cytokines mediate the differentiation of Tregs and Th17 cells [41, 42]. The current data imply that signaling through CD200R1 may be one important influence on these pathways of T-cell differentiation. Increased signaling through CD200R1 may bias toward Tregs and away from Th17 cells, and thus may be beneficial in SLE.
Downregulation of CD200R1 in SLE may contribute to impaired generation of regulatory signals, and increased production of CD200 in vivo could bind to other receptors such as CD200R2 to CDR200R4 [14], thereby transmitting stimulatory signals leading to the enhanced differentiation of Th17 cells, as has been reported [43]. Moreover, it has been reported that CD200 engagement of CD200R1 could induce tolerogenic DCs, which in turn could promote differentiation of Tregs [39, 44, 45].
In our study, however, experiments were carried out with purified T cells, making this a less probable explanation for the findings. CD200R1 expression by DCs was also downregulated in SLE, however, suggesting that reduced generation of tolerogenic DCs in the context of decreased Tregs could contribute to unregulated development of Th17 cells.
Taking the results together, we have demonstrated in SLE patients that the number of CD200+ cells as well as the serum level of CD200 were significantly higher than in HCs, whereas CD200R1 expression was significantly lower than in HCs, especially in CD4+ T cells and DCs. In addition, in SLE patients, exogenous CD200Fc reduced the proportion of Th17 cells and rescued the defective generation of CD4+CD25highFoxP3+ T cells, whereas anti-CD200R1 antibody promoted anti-CD3/CD28-induced CD4+ T-cell proliferation. Moreover, we found that CD200 on early apoptotic cells was increased in SLE patients and may serve to limit their binding and phagocytosis by DCs. These data collectively indicate that CD200 and CD200R1 expression and function are abnormal in SLE and may contribute to the immunologic abnormalities in this autoimmune disease.
CD200 receptor 1
dendritic cell
downstream of tyrosine kinase 2
- dsDNA:
double-stranded DNA
enzyme-linked immunosorbent assay
fetal calf serum
granulocyte-macrophage colony-stimulating factor
healthy control
- mAb:
monoclonal antibody
peripheral blood mononuclear cells
phosphate-buffered saline
propidium iodide
regulated on activation, normal T-cell expressed and secreted
systemic lupus erythematosus
- TGF-β:
transforming growth factor beta
- Th17:
T-helper type 17
tumor necrosis factor
- Treg:
regulatory T cell.
Rahman A, Isenberg DA: Systemic lupus erythematosus. N Engl J Med. 2008, 358: 929-939. 10.1056/NEJMra071297.
Gaipl US, Munoz LE, Grossmayer G, Lauber K, Franz S, Sarter K, Voll RE, Winkler T, Kuhn A, Kalden J, Kern P, Herrmann M: Clearance deficiency and systemic lupus erythematosus (SLE). J Autoimmun. 2007, 28: 114-121. 10.1016/j.jaut.2007.02.005.
Herrmann M, Voll RE, Zoller OM, Hagenhofer M, Ponner BB, Kalden JR: Impaired phagocytosis of apoptotic cell material by monocyte-derived macrophages from patients with systemic lupus erythematosus. Arthritis Rheum. 1998, 41: 1241-1250. 10.1002/1529-0131(199807)41:7<1241::AID-ART15>3.0.CO;2-H.
Emlen W, Niebur J, Kadera R: Accelerated in vitro apoptosis of lymphocytes from patients with systemic lupus erythematosus. J Immunol. 1994, 152: 3685-3692.
Perniok A, Wedekind F, Herrmann M, Specker C, Schneider M: High levels of circulating early apoptic peripheral blood mononuclear cells in systemic lupus erythematosus. Lupus. 1998, 7: 113-118. 10.1191/096120398678919804.
Baumann I, Kolowos W, Voll RE, Manger B, Gaipl U, Neuhuber WL, Kirchner T, Kalden JR, Herrmann M: Impaired uptake of apoptotic cells into tingible body macrophages in germinal centers of patients with systemic lupus erythematosus. Arthritis Rheum. 2002, 46: 191-201. 10.1002/1529-0131(200201)46:1<191::AID-ART10027>3.0.CO;2-K.
Sakaguchi S, Wing K, Miyara M: Regulatory T cells - a brief history and perspective. Eur J Immunol. 2007, 37 (Suppl 1): S116-S123.
Sakaguchi S: Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol. 2005, 6: 345-352.
Horwitz DA: Regulatory T cells in systemic lupus erythematosus: past, present and future. Arthritis Res Ther. 2008, 10: 227-10.1186/ar2511.
Ursaciuc C, Surcel M, Ciotaru D, Dobre M, Pirvu IR, Munteanu AN, Alecu M, Huica R: Regulatory T cells and TH1/TH2 cytokines as immunodiagnosis keys in systemic autoimmune diseases. Roum Arch Microbiol Immunol. 2010, 69: 79-84.
Webb M, Barclay AN: Localisation of the MRC OX-2 glycoprotein on the surfaces of neurones. J Neurochem. 1984, 43: 1061-1067. 10.1111/j.1471-4159.1984.tb12844.x.
Barclay AN, Ward HA: Purification and chemical characterisation of membrane glycoproteins from rat thymocytes and brain, recognised by monoclonal antibody MRC OX 2. Eur J Biochem. 1982, 129: 447-458. 10.1111/j.1432-1033.1982.tb07070.x.
Bukovsky A, Presl J, Zidovsky J, Mancal P: The localization of Thy-1.1, MRC OX 2 and Ia antigens in the rat ovary and fallopian tube. Immunology. 1983, 48: 587-596.
Gorczynski R, Chen Z, Kai Y, Lee L, Wong S, Marsden PA: CD200 is a ligand for all members of the CD200R family of immunoregulatory molecules. J Immunol. 2004, 172: 7744-7749.
Rijkers ES, de Ruiter T, Baridi A, Veninga H, Hoek RM, Meyaard L: The inhibitory CD200R is differentially expressed on human and mouse T and B lymphocytes. Mol Immunol. 2008, 45: 1126-1135. 10.1016/j.molimm.2007.07.013.
Wright GJ, Cherwinski H, Foster-Cuevas M, Brooke G, Puklavec MJ, Bigler M, Song Y, Jenmalm M, Gorman D, McClanahan T, Liu MR, Brown MH, Sedgwick JD, Phillips JH, Barclay AN: Characterization of the CD200 receptor family in mice and humans and their interactions with CD200. J Immunol. 2003, 171: 3034-3046.
Cherwinski HM, Murphy CA, Joyce BL, Bigler ME, Song YS, Zurawski SM, Moshrefi MM, Gorman DM, Miller KL, Zhang S, Sedgwick JD, Phillips JH: The CD200 receptor is a novel and potent regulator of murine and human mast cell function. J Immunol. 2005, 174: 1348-1356.
Hoek RM, Ruuls SR, Murphy CA, Wright GJ, Goddard R, Zurawski SM, Blom B, Homola ME, Streit WJ, Brown MH, Barclay AN, Sedgwick JD: Down-regulation of the macrophage lineage through interaction with OX2 (CD200). Science. 2000, 290: 1768-1771.
Broderick C, Hoek RM, Forrester JV, Liversidge J, Sedgwick JD, Dick AD: Constitutive retinal CD200 expression regulates resident microglia and activation state of inflammatory cells during experimental autoimmune uveoretinitis. Am J Pathol. 2002, 161: 1669-1677. 10.1016/S0002-9440(10)64444-6.
Rosenblum MD, Olasz EB, Yancey KB, Woodliff JE, Lazarova Z, Gerber KA, Truitt RL: Expression of CD200 on epithelial cells of the murine hair follicle: a role in tissue-specific immune tolerance?. J Invest Dermatol. 2004, 123: 880-887. 10.1111/j.0022-202X.2004.23461.x.
Gorczynski RM, Chen Z, Diao J, Khatri I, Wong K, Yu K, Behnke J: Breast cancer cell CD200 expression regulates immune response to EMT6 tumor cells in mice. Breast Cancer Res Treat. 2010, 123: 405-415. 10.1007/s10549-009-0667-8.
Moreaux J, Veyrune JL, Reme T, De Vos J, Klein B: CD200: a putative therapeutic target in cancer. Biochem Biophys Res Commun. 2008, 366: 117-122. 10.1016/j.bbrc.2007.11.103.
Kawai H, Yamada Y, Tatsuka M, Niwa O, Yamamoto K, Suzuki F: Down-regulation of nuclear factor kappaB is required for p53-dependent apoptosis in X-ray-irradiated mouse lymphoma cells and thymocytes. Cancer Res. 1999, 59: 6038-6041.
Chrest FJ, Buchholz MA, Kim YH, Kwon TK, Nordin AA: Identification and quantitation of apoptotic cells following anti-CD3 activation of murine G0 T cells. Cytometry. 1993, 14: 883-890. 10.1002/cyto.990140806.
Darzynkiewicz Z, Bedner E, Smolewski P: Flow cytometry in analysis of cell cycle and apoptosis. Semin Hematol. 2001, 38: 179-193. 10.1016/S0037-1963(01)90051-4.
Mihrshahi R, Barclay AN, Brown MH: Essential roles for Dok2 and RasGAP in CD200 receptor-mediated regulation of human myeloid cells. J Immunol. 2009, 83: 4879-4886.
Gorczynski RM, Cattral MS, Chen Z, Hu J, Lei J, Min WP, Yu G, Ni J: An immunoadhesin incorporating the molecule OX-2 is a potent immunosuppressant that prolongs allo- and xenograft survival. J Immunol. 1999, 163: 1654-1660.
Gorczynski RM, Chen Z, Yu K, Hu J: CD200 immunoadhesin suppresses collagen-induced arthritis in mice. Clin Immunol. 2001, 101: 328-334. 10.1006/clim.2001.5117.
Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM: Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med. 2003, 198: 1875-1886. 10.1084/jem.20030152.
Ren Y, Tang J, Mok MY, Chan AW, Wu A, Lau CS: Increased apoptotic neutrophils and macrophages and impaired macrophage phagocytic clearance of apoptotic neutrophils in systemic lupus erythematosus. Arthritis Rheum. 2003, 48: 2888-2897. 10.1002/art.11237.
Fransen JH, van der Vlag J, Ruben J, Adema GJ, Berden JH, Hilbrands LB: The role of dendritic cells in the pathogenesis of systemic lupus erythematosus. Arthritis Res Ther. 2010, 12: 207-10.1186/ar2966.
Jin O, Sun LY, Zhou KX, Zhang XS, Feng XB, Mok MY, Lau CS: Lymphocyte apoptosis and macrophage function: correlation with disease activity in systemic lupus erythematosus. Clin Rheumatol. 2005, 24: 107-110. 10.1007/s10067-004-0972-x.
Courtney PA, Crockard AD, Williamson K, Irvine AE, Kennedy RJ, Bell AL: Increased apoptotic peripheral blood neutrophils in systemic lupus erythematosus: relations with disease activity, antibodies to double stranded DNA, and neutropenia. Ann Rheum Dis. 1999, 58: 309-314. 10.1136/ard.58.5.309.
Smed-Sorensen A, Lore K, Walther-Jallow L, Andersson J, Spetz AL: HIV-1-infected dendritic cells up-regulate cell surface markers but fail to produce IL-12 p70 in response to CD40 ligand stimulation. Blood. 2004, 104: 2810-2817. 10.1182/blood-2003-07-2314.
Jenmalm MC, Cherwinski H, Bowman EP, Phillips JH, Sedgwick JD: Regulation of myeloid cell function through the CD200 receptor. J Immunol. 2006, 176: 191-199.
Ravichandran KS, Lorenz U: Engulfment of apoptotic cells: signals for a good meal. Nat Rev Immunol. 2007, 7: 964-974. 10.1038/nri2214.
Petermann KB, Rozenberg GI, Zedek D, Groben P, McKinnon K, Buehler C, Kim WY, Shields JM, Penland S, Bear JE, Thomas NE, Serody JS, Sharpless NE: CD200 is induced by ERK and is a potential therapeutic target in melanoma. J Clin Invest. 2007, 117: 3922-3929.
Pallasch CP, Ulbrich S, Brinker R, Hallek M, Uger RA, Wendtner CM: Disruption of T cell suppression in chronic lymphocytic leukemia by CD200 blockade. Leuk Res. 2009, 33: 460-464. 10.1016/j.leukres.2008.08.021.
Gorczynski RM, Lee L, Boudakov I: Augmented induction of CD4+CD25+ Treg using monoclonal antibodies to CD200R. Transplantation. 2005, 79: 1180-1183. 10.1097/01.TP.0000152118.51622.F9.
Kohut E, Hajdu M, Gergely P, Gopcsa L, Kilian K, Paloczi K, Kopper L, Sebestyen A: Expression of TGFβ1 and its signaling components by peripheral lymphocytes in systemic lupus erythematosus. Pathol Oncol Res. 2009, 15: 251-256. 10.1007/s12253-008-9119-8.
Zhou L, Chong MM, Littman DR: Plasticity of CD4+ T cell lineage differentiation. Immunity. 2009, 30: 646-655. 10.1016/j.immuni.2009.05.001.
Zhu J, Paul WE: Heterogeneity and plasticity of T helper cells. Cell Res. 2010, 20: 4-12. 10.1038/cr.2009.138.
Voehringer D, Rosen DB, Lanier LL, Locksley RM: CD200 receptor family members represent novel DAP12-associated activating receptors on basophils and mast cells. J Biol Chem. 2004, 279: 54117-54123. 10.1074/jbc.M406997200.
Gorczynski RM: Thymocyte/splenocyte-derived CD4+CD25+Treg stimulated by anti-CD200R2 derived dendritic cells suppress mixed leukocyte cultures and skin graft rejection. Transplantation. 2006, 81: 1027-1034. 10.1097/01.tp.0000214984.65520.50.
Gorczynski RM, Chen Z, Kai Y, Wong S, Lee L: Induction of tolerance-inducing antigen-presenting cells in bone marrow cultures in vitro using monoclonal antibodies to CD200R. Transplantation. 2004, 77: 1138-1144. 10.1097/01.TP.0000121773.18476.1C.
The authors wish to thank the health professional staff from Department of Rheumatology, Peking Union Medical College Hospital and the patients for their participation in the study, and thank Dr Wanjun Chen from NIDCR, NIH, Bethesda, MD, USA for his helpful discussion and critical reading of the manuscript.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YL, L-dZ, and L-sT developed the study, analyzed the data and drafted the manuscript. S-nQ, YR, LZ, XD, YC, and Y-xW participated in the data collection and performed the data analysis. WZ, X-fZ, F-cZ, and F-lT participated in the enrollment of patients. XZ and PEL designed the study, participated in the data analysis and manuscript preparation. D-nB, WH, and X-tC participated in coordination of the study. All authors read and approved the final manuscript.
Yang Li, Li-dan Zhao, Lu-sha Tong contributed equally to this work.
Electronic supplementary material
Additional file 2: Supplementary Figure S1 presenting fluorescence-activated cell sorting (FACS) plots specifically showing CD200 expression in CD11c-CD123high plasmacytoid DCs (pDC) (Gate 2) and CD11c+CD123- myeloid DCs (mDC) (Gate 3). (DOC 522 KB)
Additional file 3: Supplementary Figure S2 showing the serum CD200 level did not correlate with the Systemic Lupus Erythematosus Disease Activity Index score, anti-dsDNA, IFNα, IL-6, or B-cell activating factor belonging to the TNF family (BAFF) in SLE patients. (DOC 291 KB)
Additional file 4: Supplementary Figure S3 showing expression of CD200R1 in naïve T cells (CD4+CD45RA+) and memory T cells (CD4+CD45RO+) of SLE patients tended to decrease compared with HCs, although it did not reach statistical significance ( P = 0.50 and 0.11, respectively). Naïve T cells had less CD200R1 expression than memory T cells both in HCs and SLE patients (P = 0.0003 and P < 0.001). (DOC 162 KB)
Additional file 5: Supplementary Figure S4 showing immunoblot analysis of the expression of DOK2 (left) and p-DOK2 (right) in CD4+ T cells. CD200Fc induced phosphorylation of DOK2 (lane 2 on right). (DOC 231 KB)
Additional file 6: Supplementary Table S2 showing the effect of CD200 signaling on the differentiation of T-helper cell subsets. (DOC 86 KB)
Additional file 7: Supplementary Figure S5 showing the proportion of CD4+CD25highFoxp3+ T cells in new-onset active untreated SLE patients was significantly lower than in HCs (median 1.42, interquartile range 0.75 to 2.43 vs. 2.79, 1.95 to 4.52; P = 0.014). All cells plotted were CD4+. (DOC 406 KB)
Authors' original submitted files for images
Below are the links to the authors' original submitted files for images.
Rights and permissions
About this article
Cite this article
Li, Y., Zhao, Ld., Tong, Ls. et al. Aberrant CD200/CD200R1 expression and function in systemic lupus erythematosus contributes to abnormal T-cell responsiveness and dendritic cell activity . Arthritis Res Ther 14, R123 (2012). https://doi.org/10.1186/ar3853
DOI: https://doi.org/10.1186/ar3853


text:
A patient at Seattle Children's Hospital has died from a mold infection.
The patient was one of six to develop an infection from 2018-2019, according to Alyse Bernal, public relations manager for the hospital.
The infections follow several operating rooms being shut down in May by the detection of Aspergillus mold in the air. The hospital said that the risk to patients was low, but that it was contacting those who might have been exposed.
The Children's Hospital patient died after developing an Aspergillus infection in 2018, Bernal said. Details about the patient and the case have not been shared for the sake of privacy.
Gaps in air filtration is believed to have been key in the presence of mold, Bernal said. While the hospital works with outside industrial hygienists to clear the rooms of Aspergillus contamination, all 14 of the hospital's main operating rooms remain closed, Bernal said.
"We are systematically implementing improvements and corrective actions, and then retesting the air to validate our efforts have been effective. It is difficult to predict when we will be able to safely resume operations but our patients' safety is our priority and we will reopen our operating rooms when we are confident they are safe for patient care," Bernal said.
Aspergillus is a common mold that most people breathe without getting sick but that poses a greater risk to those with weakened immune systems or lung disease, according to the Centers for Disease Control and Prevention. Health problems can include allergic reactions, lung infections and other organ infections.
Those who developed infections at the hospital were at an increased risk because of the type of procedure they had, Bernal said.
Mold infections in hospitals have had fatal consequences before.
Mold played a part in five deaths between October 2014 and May 2016 at two University of Pittsburgh Medical Center hospitals, according to a 2017 report.
Those patients were exposed to Mucor and Rhizopus mold.
Those who died of the infection were transplant patients. The report showed that both the hospital and the facility that handled the hospital's linens tested positive for mold.


text: Custom Track Uniforms – Choose from our wide selection of singlets, speed suits, and 2 piece compression jerseys. Get a great value on everything you need, from bags to warm up suits, with one of our team packs. And for the coaches, families, and fans, show them who you're rooting for with our coaches' gear, beanies, headbands, visors, and spirit wear. Contact Us Today!


text: Your site is currently inactive. Please login to your account to bring your site back online. If you have any questions please email firstname.lastname@example.com. If you are visiting this website and know someone that works at this organization, please let them know of this issue so that they can resolve it.


text:
Furniture / Upholstery Hotline: 01708-444-279
Kitchen / Bedroom Hotline: 01375 373 941
Monday to Saturday 9am - 5pm
Free Delivery on all items over £250*
7 Day returns policy*
Brand Price match policy*
From the planning to the installing congratulations on a wonderful job made on my kitchen and bedrooms.
You should be very proud of the work force, Alan, John, Phil, Derek, Dave and Jason each one was very polite and a joy to have in my property. I thank everybody from the bottom of my heart.- Eileen Smith-Allen
The Intotal Designs Great Baddow Cross Back Dining Chair PU Seat is part of the Great Baddow range of living room and dining room furniture. Special attention has been paid to the design and overall sizes of this range and is based upon the sucessful compact Little Baddow range with larger units and tops. This product can be mixed with the smaller Little Baddow range and therefore our comprehesive range offer the flexibility and choice to furnish even the most awkward of living spaces. All the items in our Great Baddow Oak range are constructed from hand selected knotty oak and high quality oak veneers for added strength and then hand aged to give a sense of authentic character to each timber. The solid oak is then treated by an enhancing layer of wax which not only protects the wood but also brings out the natural beauty of the grain. There is a selection of wall units sideboards dining tables and chairs and matching occasional tables which finish off this beautiful living & dining collection. If you cannot find what you are looking for or if you have any further questions please either email us or telephone our Elm Park showroom on 01708.444279.


text:
If I point a domain (www.site.com) to a subdomain (site.cool.com), can I use a $_SERVER variable to see that the user came to the subdomain from www.site.com? I don't want the site.cool.com to ever show publicly.
Can you explain what you mean by pointing a domain to subdomain? You mean redirection? And what do you mean you don't want site.cool.com to show publicly? On google? On in web browsers' location bar?
What I mean by 'pointing' is actually going to the registrar and having that domain point to that site (with the subdomain).
And yes, I do not want the site.cool.com to show publicly (just show a blank page or redirect somewhere else), on google or the location bar.
Maybe there is a better way to do this but I basically need to figure out how the flow works…do I take the domain www.site.com and point to a dedicated IP address for the server it's going to point to and then do I need to setup an A record to point to the subdomain on my end?
Sorry, but I still don't fully understand, first you say you want www.site.com to point to site.cool.com and then you say you don't want the site to be available at site.cool.com. Does it mean you want the current site at site.cool.com to load when someone browses to www.site.com? If that is the case then this means you don't need site.cool.com domain at all, you need to take the domain www.site.com and point it to your site. I don't know what site.cool.com has to do with it? It seems redundant.
Yes, you need to point www.site.com to the IP of the server but I still don't get why you would like to point it to the subdomain - you can't point a domain to a subdomain like this, the domain's purpose is to direct the browser to the actual IP address via DNS, not to another (sub)domain. I don't know why you need the subdomain if you are not going to use it…
The project is a website were users can create their own website using one of the pre-made templates and a small CMS. So each site will be located on a subdomain (like: drsnow.site.com or energyplus.site.com). Whatever sub-domain they choose. Then we want them to purchase their own domain name and point it to the sub domain. My problem is that the client doesn't want the subdomain site (energyplus.site.com for example) to show if they go there directly. They only want the website to show up if they go to the site from the domain they purchase (www.energyplus.com). I'm just not sure of the best way to handle that.
So you need to point both the subdomain and the purchased domain to the same server. What I would do is I would always point both of them to the same site, I would not turn the subdomain off because in certain cases when the main domain stops working for any reason (for example a user fails to renew the domain) then the subdomain is always there to be used. Once it's set up like this then it's just a matter of creating a redirection from the subdomain to the main domain. For Apache servers you can do it easily in a .htaccess file in each site's subdirectory or in the main Apache config file (by searching it's easy to find examples like this).
Then you can even provide users with an option to turn on and off the redirection, which would result in your system simply creating or deleting the redirection directives in the .htaccess file.


text:
From a performance at the New Victoria Theater.
Joni appears on stage with her acoustic guitar.
Members of the band and the instruments played throughout the concert:
Tom Scott on flute, saxophone, clarinet, triangle
Robben Ford on guitar
Roger Kellaway on electric piano/keyboards
Max Bennett on bass
John Guerin on drums
Log in to make a comment
cooksforest on :
This song brought me to Joni mitchell, being a wild teen, Joni and her gift of settling a spirit through her lyrics and melodies has also brought me to a very comfortable place spiritually. No one will ever be able to raise the bar, she is a great messenger among us. How lucky are we. Help me, Helped me straighten out my intimate life many times and no doubt forever.
bob1bob on :
I agree--AWESOME as they say...as young and fun as it gets but with beautiful collaboration, macho band etc. Perhaps my favorite song and version/performance of all time too...it was nice to be a teen and have fun--but yet have the greatest teacher too--Joni embodies so many disciplines of study.
disco5000 on :
exellent. my favorite song of all time. :):):)
Didi on :
Amazing....even years later. Amazing.
pero on :
On "Help Me" we can share the lighter moments of Joni`s search for real and intensive love. It feels like springtime and falling in love is as natural as the newborn world around us, getting into a fresh sunny season.
In my ears and withit in my music feeling this wonderful song never has been faded. It is unique like a blossom of a beautiful, rare flower.
PoetryMan11 on :
This has got to be the best version of Joni's biggest hit that I have ever heard. What I especially like is that the end resolves itself and doesn't just fade out like the recording does.


text:
Just In Case Mechanical, LLC has established itself as a trusted HVAC company near me for homeowners and businesses seeking expert Heating & Cooling services.
just, case, #mechanical, established, itself, trusted, hvac, company, near, homeowners, businesses, seeking, expert, heating, cool
All things Day Van related. For enthusiasts of American Day Vans, Chevy Astro, GMC Safari, Dodge, Ford, Chrysler, G20, Econolines and much more. Friendly forum for chat, advice, information, technical, mechanical data, parts, recommended services and
free, forum, american, chevrolet, astro, safari, chrysler, dodge, ford, help, dayvan, chat, parts, spares, technical, #mechanical
Higher technological Institute Mechanical Department
#mechanical, department, h.t.i, higher, technological, institute
To explain how Newton's laws of motion applies to mechanical conservation of momentum.
perpetual, motion, explain, newton's, laws, applies, #mechanical, conservation, momentum
Keep your hands and feet inside the vehicle at all times, and have a nice stay at Crazy2nd's forums
#mechanical, frost, keep, your, hands, feet, inside, vehicle, times, have, nice, stay, crazy2nd's, forums
A collection of custom designs, design resources and downloads for websites, photographers, designers, coders, business owners, anime lovers, technical engineers and motion graphics enthusiast.
coding, game, develop, publishing, photography, metals, css/html, splash, page, themes, games, intro, xbox, robotics, design, development, posters, gaming, stationery, #mechanical, engineering, world, news, buttons, logos
Free forum : MechTech the MMO Development Forum. Free forum : Mechanical Technologys
free, #mechanical, technologys
This website include all these materials which relate to industry are such as Process Mechanical Equipment I, Process, Instrumentation & Control I Industrial Chemistry, Applied Fluid Mechanics, Pneuma
process, operation, technology, petrochemical, proces, imco, students
Free forum :Suzuki Marauder GZ125 Owners discussion forum, feel free to join in, no holds barred marauder action, the slow cruiser. Hints and tips, and mechanical advice.
forum, suzuki, marauder125, marauder, gz125, owners, discussion, cruiser
Free forum : to all 2004 upm mechanical student. Free forum : Mechanical & Manufacturing UPM 2004
free, #mechanical, &, manufacturing, 2004
Free forum : EMS, A Vendetta Online Guild
Free forum : A completely free role playing site based on a digital war between humans and V-Viruses! -Temporarily using a Matrix theme until I can design one myself later-
free, ritshand, digital, virus, device, chips, cyber, machine, mecha, #mechanical
RMboards. com and RMboards. net
rmboards.net, message, boards, rmboards, rmboards.com, restoration, #mechanical, wind, fire, water
This forum for UM mechanical student only. UM Mechanical Engineering Forum
ummechanical, #mechanical, engineering, forum
leedsmecheng - The LUU Mechanical Engineering Society
leedsmecheng, leeds, #mechanical, engineering, university
For All Your Custom Needs. AOC Automotive. aoc aocauto mechanics mechanical tips tricks audio subs amps troubleshoot audio help with audio automotive aocforum auto forum cars turbo forumaoc aocauto ro
aocauto, mechanics, #mechanical, tips, tricks, audio, subs, amps, troubleshoot, help, with, automotive, cars, turbo, forumaoc, rockauto
Place for PJB Mechanical students to make revision and references
student's, reference, place, #mechanical, students, make, revision, references
A Discussion among the skilled trades.
contractors, skilled, trades, electrican, plumber, #mechanical, forum, construction, leads, contractor, advertising, plumbing, electrician, hvac, jobs, technician


text:
No great words of wisdom this week. For those who celebrate, we're at the end of Holy Week, today being Good Friday and Sunday being Easter. So here is a nice assortment of links for anyone who feels like checking them out. Whatever your beliefs, I wish you a wonderful weekend and a bit of whatever makes you smile, whether that's writing time, a good book, family celebrations, or a bit of everything. Enjoy!
A Brief Literary History of Robots – Mostly because I couldn't resist the first image, but also for the fun reading list.
George Saunders: What Writers Really Do When They Write – One writer's take on process.
Don't Be a Dick: Colum McCann's Advice for Young Writers – Some great tips, delivered with humor.
Julie Ann Walker: "I'm a Feminist and I Write Romance" – A smart look at why these two things are not mutually exclusive.
NPR on Tumblr – Some great vintage images shared in honor of National Library Week.
Salinger's Nightmare – An unemployed actor's attempt to acquire permission to adapt The Catcher in the Rye.
Kurt Vonnegut's Greatest Writing Advice – Tips that still shine, ten years after the author's death.