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If you're the proud owner of a Jeep vehicle, you're most likely also benefiting from the low cost of insuring one. Three of the least expensive cars to insure are all made by Jeep, according to a recent study by Insure.com, an independent consumer insurance website.
Lincoln is in the middle of reinventing itself, along with both Cadillac and Chrysler. All three American luxury brands are upping their efforts to deliver cars that can go toe-to-toe with the best that Germany has to offer. Cadillac is bringing an all-new flagship to the 2015 New York International Auto Show - and now it appears that Lincoln is, too. It's the 2016 Lincoln Continental Concept, and it looks astounding.
One of the aspects of driverless cars that you might not have taken into consideration is that they will be consistently, frustratingly, law-abiding. No more blowing through stop signs (not even rolling stops), no more illegal right turns on red, and definitely no speeding. Ever. But thanks to Ford, the super annoying aspects of robotic cars can be yours now, at least if you live in Europe.
You can trace the history of the Mercedes-Benz M-Class visually; many design cues, like its wraparound rear window, have remained on the vehicle across multiple generations. That said, the current M-Class is getting a bit old in two important ways - its looks, and its name. As Mercedes-Benz moves to a sleeker front end and a new naming scheme, the brand rolled all those changes into one, and now we have ourselves the new Mercedes-Benz GLE-Class.
In the interest of full disclosure, you should be aware of two things before you read this article. First, Web2Carz offers services to connect our readers to auto lenders. We keep a firm wall between our editorial content and our business operations, but we also can't claim to be totally disinterested in this topic.
It's no surprise that buyers often compare both new and used cars. For a given price point, the market for potential cars explodes once you start looking away from new cars alone. Yes, you might give up the warranty, but if you're looking for something unique, used cars provide a good value ... provided you pick the right one. Bang for Your Buck takes a look at five used cars you could get with the money you'd spend on the new car we've tested recently.
Nighttime visibility remains an issue that we haven't fully sorted out yet. Sure, we have brights for when it gets really dark, but those brights can over-illuminate parts of the road or, worse, blind oncoming traffic. There has to be a better way to light up everything important, right? That's what GM's European brands believe, and they're hard at work on a lighting technology that uses your eye movements to figure out what needs to be lit up.
It is no less than an exciting week in the world of car videos - from the serious and precautionary to the downright fun. From Dallas to Dubai and back again, Web2Carz brings you another installment in our weekly Top Five (truly) Kick-Ass Car Videos.
There's a very good chance you've owned a car at one point that had a spoiler. There's an equally good chance you never bothered to really look at it, perched on the rear trunk lid like some useless sixth digit.
Do you know why that car had one? Or why cars continue to employ these accessories in their design schemes? Here's a brief history of the spoiler and an explanation as to why they're pointless.
In areas with carpool lanes, it can be very frustrating to see open lanes when the rest of the traffic is going at a crawl. This causes people to try many creative solutions (anything short of actually carpooling) to get away with driving solo in the high-occupancy vehicle (HOV) lane.
The electric bicycle market is steadily growing, and as battery technology continues to advance, the quality of the bikes continues to increase. So who better to get into the e-bike game than the battery engineer team responsible for the world's most advanced hybrid supercar? When Rimac Automobili teamed up with Trefecta Mobility to design a military-grade super e-bike the gauntlet had officially been thrown down.
It doesn't matter how much money a person makes; everybody loves a good deal. That's even true with high-end sports cars. Take Jaguar for instance; for the F-Type's 2016 model year, the automaker is rolling out a slew of new standard offers, covering everything from warranty periods to trim levels to standard equipment. It all combines together to produce an F-Type that's better than ever before, and it lands at dealerships as early as April.
For a few years there's been talk, in certain extremely small circles, of a coming e-bike revolution. The future of transportation, they say, is about to change radically as the population explodes and more people live in densely populated cities. And that much is true, but that doesn't mean that e-bikes are poised to take over. For one thing, no one can seem to agree on what an e-bike is.
If you've been living under a rock for the last day or two, you might have missed the announcement that Broken Lizard is attempting to crowd-fund its next venture, Super Troopers 2. Those who chose to donate early on (they've already surpassed the $2M minimum required to make a bare-bones movie) had a variety of perks come along with that donation. One lucky patron of the arts plunked down $35,000 for the chance to take home the "squad car" they'll be using in the movie.


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Recommendations of The Canadian Council for Public-Private Partnerships presented to the Walkerton Inquiry Part 2: Public Meeting Management of Water Providers September 20, 2001 Presentation by: Hon. Michael H. Wilson, Chair, CCPPP The Canadian Council for Public - Private Partnerships (CCPPP) Interest in the Walkerton Inquiry First and foremost, CCPPP has an interest in seeing infrastructure and service investments keep pace with the needs of its citizens. The tragedy in Walkerton in 2000 has focused our collective attention on the state of water related infrastructure in this province and on the methods of delivery. CCPPP's first interest is ensuring that all options are explored in that regard. Secondly, we recognize that it is an increasingly globalized environment within which all Canadians live and that for Canada to be competitive, it must constantly upgrade its public services and infrastructure to attract business investments. CCPPP believes that the capacity of the public sector to meet the accumulative infrastructure requirements in all sectors is limited and that the instrument of public-private partnerships is one method of bridging the gap between public sector capacity and the demands being placed upon it. Our position is reinforced by our exposure to other government-led programs, most notably the United Kingdom, Australia and New Zealand and increasingly in the EU countries which support the concept of public-private partnerships and the modernization of their infrastructure through a variety of such arrangements. South Africa is also well into the development of a similar program. CCPPP's intention is to bring forward to the Inquiry options in the provision of water services to those traditionally offered directly by municipal organizations either through public departments or agencies and public utility commissions. The nature of the arrangements that we support are public-private partnerships rather than privatization. Our Mandate The Canadian Council for Public-Private Partnerships is supported by membership fees and revenues generated through events and research publications. No direct government grants are received to support our operations although research activity is often undertaken through partnerships with government organizations. CCPPP works on a national platform and draws members in almost equal numbers from the public and private sectors. We work very closely with governments across Canada and support them through the provision of expert advice and research materials. The CCPPP Board of Directors is made up both of public and private senior representatives as well as union representation. With interest in the particular issues before the commission would be our members representing the following: • Municipalities, some with private operators, many looking at it as an option. • Provincial governments throughout Canada • Engineering and technical consultants both to public and private clients • Private water companies • Construction companies • Management consultants working in the environmental field as advisors to both public and private sectors • Financial and legal advisors to both sectors Background CCPPP has been involved in the Inquiry in a number of ways: In January 2001, CCPPP submitted a report to the Inquiry entitled "A Submission to the Walkerton Inquiry (Part II) Concerning Alternative Models of Water Systems with Private Sector Involvement". A companion document entitled "Overview of Successful Public-Private Partnerships in the Water Sector" accompanied the submission. The former is posted on the Inquiry's website for viewing. The latter is a very important piece of research conducted by CCPPP which examined nine North American water and wastewater projects looking at financial savings to the municipalities, public satisfaction with the private sector operations taking into account such considerations as the impact on water rates, quality of service and so forth. Each project carries a testimonial from either an elected representative or a senior works commissioner with direct knowledge of the individual project's performance. This publication is not posted on the website for technical reasons but is critical to appreciating the position of CCPPP. Appearances were also made at the technical meetings of June 20 and July 5. In addition to technical advisors, CCPPP was accompanied to the meetings by Mayor Brian Murphy and City Manager Al Strang, City of Moncton (June 20 Meeting) and Mayor Deb Shewfelt, Town of Goderich (July 5), who shared the experiences of their respective communities in public-private partnerships in water delivery. In addition, CCPPP sought the input of Ofwat (Office of Water Services) in the United Kingdom to comment on the CUPE commissioned report on the UK Water Privatization recognizing that certain errors were presented within the Hall report and required correction and clarification. As the economic regulator of privatized water services in the UK, only Ofwat could accurately identify misrepresentations or misinterpretations within the report. The response provided by Director General Philip Fletcher appears on the Inquiry website as a communication between him and the Executive Director of CCPPP. Again, this is a valuable contribution to the understanding of how effectively the private sector has been involved in the provision of water services. The CCPPP position on all matters related to the development of a future water delivery system for Ontario are captured in those various documents and summaries of discussion. Our purpose by way of this submission is not to revisit the detail of those individual documents. Rather our intention is to offer some concluding thoughts based on our research and technical knowledge of private sector operations in the water field. Sound System of Water Delivery The overall objective is first and foremost to achieve a sound water delivery system, one in which all factors contributing to the achievement of that system are free from influence or conflict that could potentially negatively affect overall performance of the system. Ensuring that all the components that have to interlock are in place is the first goal: the setting of water quality standards, non-performance detection responsibilities, enforcement capabilities, etc. All of these should be in place regardless of whether a private or public model of delivery is contemplated. Within that context, we think there are a number of considerations to ensure the integrity of any system. These relate to the division of responsibility in the establishment of water quality standards & regulations, the enforcement of the standards, the financing of water systems and the elimination of conflicts with water treatment and delivery. A great deal of discussion has already taken place throughout the hearings into these matters and carefully considered by experts advising the Commission. We, therefore, want only to reinforce the position that separation of each of these activities is warranted. Integrity of the system can only be achieved when conflicts of interest are removed such that each activity functions with clear objectives unfettered by extraneous considerations achieving other unrelated goals. Water quality standards should be established to ensure safe potable water is available to our citizens. Supportable standards should not be compromised because of the recognized costs to achieve the standards. Performance of all systems should be monitored, whether public or private. Violations should be identified and violators dealt with fairly and consistently. Complications arising when the regulator is also the operator need to be eliminated. Similarly, the decision to set standards or enforce standards cannot be conflicted by the obligation to fund the upgrade to equipment or service to achieve the standards. When all these functions reside within the government or in an agency of the government, the potential for conflicts exist. These are matters outside of the mandate of our Council and therefore our comments are offered only as observations rather than recommendations. Public-Private Partnerships as an Option The Canadian Council is, however, very well positioned to comment on the benefits that we have seen when public - private partnership models for delivery are utilized. It is our intent to offer assistance to the Inquiry as it looks to solutions in creating a future sound water delivery system in this province. A commissioned paper of the Inquiry prepared by Professor David Cameron of the University of Toronto explored the relationship of ownership and operations and performance. He concluded that actual ownership as a characteristic itself appeared to be relatively unimportant in the achievement of good water quality. Rather, other factors such as "the skills and training of the staff, the overall management systems that have been put in place, the state of development of water engineering and water technologies, the condition of the infrastructure, the existence of effective monitoring capability and adequate feed-back loops" are the factors that determine the quality of water being delivered to citizens in any given jurisdiction. This finding supports much of our own research. For those matters within the scope of operations and physical assets, we believe that the achievement of a sound system is very much dependent upon the ability to finance the physical plant for water treatment and distribution systems and on the availability of highly trained staff with access to state of the art technology and innovation to operate it. The Canadian Council for Public-Private Partnerships would suggest that there are factors influencing private sector organizations that compel them to consider the above characteristics somewhat differently than a public sector organization would. Based upon our research and upon our considerations of those factors which reflect some of the influences and drivers of private versus public businesses, we believe that there is a clear role for the private sector to play in the achievement of a sound water deliver system for Ontario and we, therefore, make one recommendation to the Inquriy: RECOMMENDATIONS That the Walkerton Inquiry recognize the benefits that can be derived from public- private partnerships in the water sector through the private sector's participation in a spectrum of activities including finance, design, build and operation of water treatment facilities and distribution networks. That the Walkerton Inquiry include within its recommendations to the Government of Ontario the increased reliance on public-private partnerships as an option to public sector delivery. Financing One of the areas in which the private sector has made the greatest impact is in the actual financing of water infrastructure. As Dr. W. H. Emery, Director General of Ofwat points out in his letter (July 18, 2001) on the modernization of the British water and wastewater system, between 1990 and 1999 capital investment made by the private sector to deliver higher water quality and sewage treatment standards set both nationally and at a European level, was close to £ 33 billion (May 1999 prices). By 2005 that investment is projected to be in excess of £50 billion. This level of investment was not remotely within the ability of the public sector and most would agree that the private sector is responsible for the modernization of a deteriorated public system in that country. Within the Canadian context, we have seen the partnering of the public and private sectors solve a long time problem in Moncton, New Brunswick. Mayor Brian Murphy and City Manager Al Strang accompanied CCPPP to the June 20 Inquiry meeting to discuss their experience. For many years Moncton chlorinated its water supply but did not meet the Canadian water quality guidelines for drinking water. Boil water orders were becoming increasingly common. Citizens in random sample surveys consistently recognized low water quality as the leading municipal service issue. In 1995 they estimated a cost of $32 million to provide a solution by way of a new treatment plant. They did not have the financial capacity to undertake the project and the future did not suggest that they would any time soon. They turned to the private sector through a competitive proposal process to develop a partnership with a private sector partner. In 1999 a new plant was up and running in Moncton delivering one of the highest water quality standards in this country. For $23 million ( $11 million less than if delivered traditionally by the public sector) a private company had entered into a public - private partnership (Design Build Finance Operate) with a 20 year lease-license agreement with the full operation of the plant provided by the private sector. Water rates are established by the public sector. Regulations are imposed by the public sector. This has been such a success and solved such a critical problem for the municipality that they are currently examining the feasibility of turning the refurbishment of the aged distribution system (some parts of which are 122 years old) to the private sector with an estimated private sector investment required of $70 million. Many other examples can be drawn from across North America and Europe as developed nations and clearly public - private partnerships are a standard instrument in achieving infrastructure investment and operations in developing countries, becoming almost the exclusive method used now in World Bank projects. No comprehensive study has been completed to date to accurately reflect the infrastructure investment deficit and the future requirements in water and wastewater systems across Canada. Such a study is needed because it will help to focus the discussion. However, a study conducted by the Federation of Canadian Municipalities in 1996 estimated the investment deficit at $44 billion for water supply services and $9 billion for sewage treatment in Canada. It could be reasonably expected that these figures would be higher today. CCPPP does not believe that the public finance capability in Ontario and certainly across Canada can address this infrastructure and service investment deficit and future requirements using government sources alone. In water and wastewater services, we question the accuracy of any studies that suggest Ontario municipalities currently have the financial capacity to address this investment deficit recognizing that the full costs to do so have not yet been determined. Operations The private sector has most commonly established itself across North America as operators of water and wastewater treatment facilities. This is gaining in popularity in the USA and observers of this particular industry report that more and more municipalities are exploring public - private partnerships as an alternative to traditional public sector operations. An indication that satisfaction is being achieved is the high level of contract renewals that are being signed. Many North American municipalities have found the public - private model of delivery better meets its obligations to operate a highly efficient and technically sound water treatment and wastewater systems than their traditional model of delivery. In terms of cost efficiencies alone, estimates by consultants working in this field are reporting savings in the range of between 30% and 40% over traditional public sector deliver. Why are the efficiencies achieved by the private sector so great and how is it possible? The benefits of using the private sector are explained in detail in the CCPPP January 2001 submission to the Inquiry. However, briefly, CCPPP believes there are a number of characteristics of private sector operations, as mentioned above, that position them to focus on efficiency and risk reduction which ultimately produces sound delivery systems. The first of these characteristics relates somewhat broadly to the nature of private enterprise in general but also specifically to private water companies. Non-performing water companies fail to thrive. There is no such thing as a second chance when a water company fails to perform. They go out of business. There is no tolerance for error when it comes to the delivery of safe drinking water - either internally within the company or externally from the clients. Water companies want to survive. Everything within the company is designed to ensure that they continue to survive. And it starts at the top with their governance structures. Municipally run water and wastewater facilities are most commonly overseen by some type of body made up of political appointees or the political representatives of the municipality - through a committee structure and Council or formally, through a public utility board. They are people elected to oversee the vast number of activities being performed by their municipal organizations or boards, being experts often in no individual area. Typically those represented on the oversight bodies have no technical background in water systems. This stands in sharp contract to the management boards of private water companies. The Boards of Directors or Management Boards of larger water companies would typically have expertise from either the technical or management side of the water business. For example, one board that we examined had 5 of 9 board of directors who had come up through the ranks of the water industry prior to appointments on the board. This ensures that the oversight of the management of operations throughout their portfolio of contracts is technically able to judge the level of quality of the service being delivered. They are charged with the responsibility to ensure that the private company thrives. The point should also be made that non technical directors drawn from the fields of management and finance also add depth and balance to the technical representatives on the Board in ensuring that health, safety and environmental issues receive strong consideration. As a first consideration we would conclude that the latter model of oversight carries a higher degree of professional competence than the government model. As a result, it is positioned, from a governance perspective, to achieve a higher quality operation and greater assurance of water quality provision. The second consideration is how a private sector evaluates and manages risk versus a public sector provider. Anything that jeopardizes a private company's ability to perform is considered a risk. Inadequately trained staff, malfunctioning equipment, faulty infrastructure and so on are unacceptable risks in the business of private sector water operations. So referring back to Prof. Cameron's findings, those characteristics of systems that are more likely to ensure good quality operations, are the factors that if mismanaged create risk. Risks are minimized at every opportunity to ensure the integrity of the service being offered. Operations and standards are monitored on an on-going basis to ensure that top performance is being achieved and that the potential for failure is minimized or eliminated. (Risks in the public sector are often not given the same consideration. There is an inherent cost to any organization that assumes risk - be it public or private. Increasingly in discussions being conducted by governments looking at "value for investment" considerations in many countries, risk is often not quantified as one of the costs being borne by government and it should be. When the public sector delivers a service, either directly or through an agency of the government, risk is being assumed and should be valued and treated as an expense of operation.) Performance contracts which penalize non-performance result in financial loss. Catastrophic failure, such as feeding contaminated water into a municipal distribution network resulting in deaths, would result in bankruptcy. Because the company's very survival is based on its performance, the focus on service quality is sharpened. Consistent with the objective to reduce and minimize risks while at the same time building efficiencies into their operations, many private water companies invest heavily in research and development activities which bring improvements on a technical and operational level to all operations throughout the network of contracts worldwide. Mr Deb Shewfelt, Mayor of Goderich, Ontario accompanied CCPPP to the July 5, 2001 Technical meeting of the Inquiry and made the point that when his municipality articulated what it felt it needed to ensure that high quality water would be available for its citizens, access to the best technology and research available rated very high on their list. Running the municipal water system themselves meant operating in isolation without the benefit of immediate access to new technologies and to new technical information. Many of the larger water companies have that capacity within their organizations. These two sets of factors, the nature of enterprise and the management of risk through all factors including governance, research and technology, training of staff, and so on, in combination compel private sector operations somewhat differently than public sector operations and bring benefits to municipalities that have selected private sector partners. This is not to say that other factors, for example political accountability*, are not a compelling driver to municipally operated systems but rather that the factors that drive private companies are perhaps more transparent in as much as their objectives are extremely clear - to thrive, to maintain a reputation as a high level performance company from the users' perspective, to establish a growing client base based on past performance and to get a return on an investment. Clarification of what a private operation means to the municipality There is a great deal of misinformation about what it means to involve a private sector company in a municipal operation. The following clarifies some of the more common myths that are raised by opponents of PPP. *It should be noted that Mayor Shewfelt of Goderich made a representation before the Technical Hearings of the Inquiry on July 5, 2001 and submitted that political accountability is in no way diminished when contracting a service provider over delivering the service directly. Water Rates Let us examine what an operating contract is versus what it is not. First, it does not take away any ability of the public sector to set the water rates. That responsibility falls within the domain of the public sector and resides there as their sole responsibility. On a long term contract, water rates may well be determined for the entire length of the contract and therefore provide predictable long term certainty about the costs being allocated to each household barring any unforeseeable circumstances. They are established by the public sector and politically determined by the public sector. Ownership of the Water Related Assets Secondly, a public - private partnership (as opposed to the privatization model) does not take the ownership of the facility out of the hands of the public sector. Most commonly in North American models, the public sector continues to own the physical asset and depending upon the terms of the contract, determine in partnership with the private sector the level of investment in the capital plant and technology to keep the plant in a good state of repair. In some contracts, this would be the responsibility of the private sector and the contract would specify the performance standard to be met on the condition of the plant. Impact on Existing Labour Force Thirdly, it does not mean that all the municipal / public - utility staff currently operating the plant would be fired and that the private operator would bring in staff from outside to operate the plant. Logic would dictate and practice would confirm that the people who best know the plants are the ones who currently operate them. Efficiencies come through increased training, technological improvements, procurement practices, etc. The protocol in the industry is to transition the bulk of staff (in many cases all of the staff) over to the private sector organization maintaining similar benefit packages to those offered by the public sector and often maintaining the pension plan through arrangements made with the public pension system such as OMERS in Ontario. Labour has not proven to be the source of the savings achieved in most operations in North America. Related to this is the misunderstanding voiced by a number of union participants at the technical meetings that the operator brings in people to staff the plant from outside therefore suggesting that these people are not people "from the community - not your neighbours". This is a misunderstanding. The private sector would have no reason to import people from outside, nor would they be likely to want to incur the expense of relocations. The plant workers live in the community. Chances are the majority of them are the same people who worked in the plant when it was a publicly operated facility. To suggest that "outsiders" take over and therefore are not committed to the plant is erroneous in all respects. Control of the Water Delivery System Thirdly, opponents of public-private partnerships in water operations would suggest that there is a loss of control to the municipality when a private operator takes over. This is not factual. Mayor Shewfelt explained in detail his council's considerations in this regard before entering into a long term operating contract with a private supplier for the Goderich system. He concluded by saying that in fact he believed that there is more control using a private sector operator than a public operator because the contractual agreement between the supplier and the provider specifies the exact expectation of the municipality on the private sector, something that would not be articulated as well with your own staff. Furthermore, penalty provisions are outlined in detail should a supplier not perform at the level specified within the contract. Again, such clear guidelines do not exist with an in-house provider. Non-performance remedies, in any case even if they were contemplated to apply to a public operator, would fall squarely back on the same municipality. Many would suggest that the existence of a clearly written contract with a clear set of penalties for non - performance make private sector operations more accountable than public systems and more transparent. Free Trade Issues Opponents of PPP have recently made a concerted effort to distort the terms of international trade agreements to suggest that the contracting of private operators in this sector (investors in the language of the agreements) can impact a municipality (sub-national in the language of the agreements) so that they lose control of their ability to manage the contract, to make changes to conditions in the terms under which the contractor operates, to make changes in the standards (for example water quality standards provincially determined), etc. A separate submission to the Inquiry will be made on behalf of CCPPP in this regard. Suffice to say that CCPPP has been in extensive discussions with the Department of Foreign Affairs and International Trade (DFAIT), Industry Canada, numerous law firms with international trade agreement expertise and concludes that there is no substantive evidence to give credence to these allegations. They distort the intent of the agreements (specifically the intent of Chapter ll of the North America Free Trade Agreement) and endeavor to create an atmosphere of uncertainty in doing business with international suppliers. Based on all information and expert advice being received by CCPPP, these assertions should be recognized for their true intent. DFAIT has embarked on a cross Canada tour of meetings with the municipal level to clarify the agreements and to satisfy concerns that may have been raised by the opponents of free trade. Conclusions There have been demonstrable successes in the private delivery of water throughout the world and more relevantly in North America. These partnerships can take on a number of characteristics depending on the needs of the region or community. In the case that the community requires improvements or development of infrastructure (plant, distribution system, etc.) and lacks the financial capacity to address the problem, the private sector can participate as a financing partner. This in combination with Design/Build expertise has proven to deliver infrastructure well under typical costs achieved by the public sector through traditional procurement. When the objective of the public sector partner is to ensure the quality of operation, the private sector can act as the systems operator within the publicly administered system. The various models are outlined on pages 4 through 7 in our submission dated January 30, 2001 which is posted on the Walkerton Inquiry website. The Canadian Council for Public-Private Partnerships encourages those charged with the responsibility to look to improvements in the future model of service delivery in water in Ontario to consider the greater use of public - private sector models for the benefits that they have proven to bring in financing, design, build and operations.
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Celebrate Good TimesIt's been all about awards, accolades and achievements this week. We've had wins and top placings in many different events and a packed house at our club presentation evening. It was great to reflect on the successes of last year and … Continue reading »
Run, run as fast as you canWe could be forgiven to think that the weekend of Mothering Sunday would be a quiet one. Not at all! There's lots to find out from Junior cross-country to fells and roads. This week's compilation is care of Irene Roche. … Continue reading »
Tiptoe through the tulips...Well, it may be a tad early for the tulips but this week you have been fell running at Flower Scar, touring Pendle on the Stan Bradshaw Pendle Round, Doddling with Daffodils, Rolling at Roddlesworth, finishing the cross country season in Lancaster, … Continue reading »
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The Alumni Association is pleased to announce this year's Reunion 2015 Key Note Speaker is Apollo 13 Astronaut Fred Haise! Make plans now to attend to his lecture on Saturday, July 11.
A HUGE thank you to Scott Bittler '83 for returning to campus and teaching our students how to get on track to financial success!
David Champoux '11 Traffic Engineer with Wolverton & Associates, Inc.
Join fellow Clarkson alumni from the Capital District Chapter at Albany Distilling Company to enjoy an hour long tour and whisky tasting, followed by a dinner at the Pump Station restaurant.
20 locations across the United States will participate on our annual service day: Clarkson Serves 2015. Check if there is a location near you and join us!
Join Clarkson Alumni around the country along with alumni and students on campus for Clarkson Serves!! Help us reach our goal of alumni and students nationwide participating together in service. * Time Change: Clarkson Campus clean up will take place at 2 pm*


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Drake may be known as being the saddest dude in the game, but not many can resist funny-man Will Ferrell. The two met up at the premiere of Ferrell's new movie 'Get Hard' and Drake can't contain his giggles.
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The DJ reveals why he got sick of traditional radio–and why he wanted to change things, fast. 'DJ SKEE Ignited' premieres tonight at 11/10c!
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It's no surprise the comedian named her book 'Have You Seen My Pants?'
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The actor reveals the awkward moment he shared at a hotel after getting the news that he would play Penguin on the hit Fox series
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Plus, the actor shows 'White Guy Talk Show' hosts Grace and Choksi how to nail the infamous villain's trademark limp
March 25, 2015
A pop-punk collaboration of such epic proportions should have made a bigger splash
Jeff Benjamin / March 26, 2015
Daily photo of your favorite musicians... being your favorite musicians
Fuse Staff / March 02, 2015
Into pop culture but think you're too old for the annual Nickelodeon event? Guess again! Here's everything you need to know
Maria Sherman / March 25, 2015


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The word pistil (from Latin pistillum meaning pestle) is also sometimes used to describe each discrete unit of the gynoecium. A pistil can consist of either a single carpel (in a monocarpous or apocarpous gynoecium), in which case it is called a simple pistil, or of several fused carpels (in a syncarpous gynoecium), in which case it is called a compound pistil.The style and stigma of the flower are involved in most types of self incompatibility reactions. Self incompatibility, if present, prevents fertilization by pollen from the same plant or from genetically similar plants, and ensures outcrossing. Stigmas can vary from long and slender to globe shaped to feathery. The stigma is the receptive tip of the carpel, which receives pollen at...
I am Odon Czintos photographer, I am member of the MAFOSZ (National Federation of Hungarian Photographers). I am interested in taking pictures since 1984. I always intend to show new faces of nature and the world surrounding us. I'm not interested only in nature photography. When something captures my mind, I try to capture it with my best knowledge and creativity. I took part in several Hungarian and international photo competition with different success. My photographs participated in joint and individual exhibitions. My photos received first, second, third places or special awards. My photograps are published in photo magazines and books. I am mostly proud of my photographs exhibited in the European Parliament and...
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Blue Moon Ice Cream
Made This Recipe? Add Your Photo
Total Time
Prep1 hr
Cook4 hrs
A lovely looking recipe posted in response to a recipe request. Cook time is freezing time.
Make and share this Blue Moon Ice Cream recipe from Food.com.
- 709.77 ml finely chopped pineapple
- 118.29 ml sugar
- 118.29 ml blue curacao, to taste
- 1892.0 ml good quality vanilla ice cream
- In a large saucepan, combine fruit, sugar and curacao.
- Bring to a boil over moderate heat, stirring until sugar dissolves.
- Simmer for approximately five minutes.
- Strain into a bowl, reserving pineapple solids.
- Temper ice cream in the refrigerator until slightly softened.
- Add pineapple and stir to combine completely.
- Return to freezer for two hours; stir again.
- Freeze until completely firm.
- Ice cream will keep approximately one week.


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Written on: 14/07/2008 by Utter Bunkum (1 review written)
Good Points
Easy and accurate imports,
Bad Points
Can't do an automatic update it seems
General Comments
I have this installed on Vista Home Premium and can use it with no problems!
The only thing I'd change is to be able to do automatic updates! Can someone suggest where I can go do manually download the update and install them?
Well recommend DMC Cross Stitch Pattern Design for Arts and Crafts Software.
As rated by real users
" Great initiative " Read More
Written by ChelseaParker
" Tried the rest... this is the best " Read More
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Narrow your search
Sprint, AT&T and other carriers have lined up with a wide range of pricing options for consumers. Samsung has a lot riding on getting off to a good start.
CNET's Marguerite Reardon takes a look at T-Mobile's new plan to entice wireless customers to jump ship from their existing carriers.
In a segment on his show, sponsored by T-Mobile US, Colbert suggests that its phones be inserted up rear ends to alleviate constipation. Now that's a product endorsement.
T-Mobile's version of the flagship smartphone comes with a year's access to the streaming video service.
The carrier is also guaranteeing that prices won't increase for customers on existing plans -- even if you're on a promotional program.
"I wouldn't rule it out," John Legere says about emulating Sprint's leasing model, but adds that there are no plans to roll one out in the near future.
The US carrier blocked its workers from organizing or even discussing problems at work and has been ordered to revise its policies.
Prices for each line range between $10 and $16 depending on the number of accounts. T-Mobile is also bundling a free domain and Microsoft Office 365 services for any company paying for additional data.
Exactly how much will Samsung's new smartphones cost? We break it down for you.
John Legere, leader of the third-largest wireless carrier in the US, is now calling on the US to change the way the auction is run.


text:
Jens V. Stein1, Marta López-Fraga1, Fernando A. Elustondo2, Carla E. Carvalho-Pinto1, Dolores Rodríguez2, Ruth Gómez-Caro1, Joan de Jong3, Carlos Martínez-A1, Jan Paul Medema3 and Michael Hahne1
1Department of Immunology and Oncology, Centro Nacional de Biotecnología, Madrid, Spain2
Departamento de Biologia Celular y Molecular, Centro Nacional de Biotecnología, Madrid, Spain3
Department of Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands
Address correspondence to: Michael Hahne, Department of Immunology and Oncology, Centro Nacional de Biotecnología, Campus de Cantoblanco, Universidad Autónoma, E-28049 Madrid, Spain. Phone: 34-91-585-4659; Fax: 34-91-372-0493; E-mail: firstname.lastname@example.com.
Published June 15, 2002Submitted: January 11,
2002; Accepted: May 14,
The TNF-like ligands APRIL and BLyS are close relatives and share the capacity to bind the receptors TACI and BCMA. BLyS has been shown to play an important role in B cell homeostasis and autoimmunity, but the biological role of APRIL remains less well defined. Analysis of T cells revealed an activation-dependent increase in APRIL mRNA expression. We therefore generated mice expressing APRIL as a transgene in T cells. These mice appeared normal and showed no signs of B cell hyperplasia. Transgenic T cells revealed a greatly enhanced survival in vitro as well as enhanced survival of staphylococcal enterotoxin B–reactive CD4+ T cells in vivo, which both directly correlate with elevated Bcl-2 levels. Analysis of humoral responses to T cell–dependent antigens in the transgenic mice indicated that APRIL affects only IgM but not IgG responses. In contrast, T cell–independent type 2 (TI-2) humoral response was enhanced in APRIL transgenic mice. As TACI was previously reported to be indispensable for TI-2 antibody formation, these results suggest a role for APRIL/TACI interactions in the generation of this response. Taken together, our data indicate that APRIL is involved in the induction and/or maintenance of T and B cell responses.
The TNF family has an important role in inducing various biological responses such as cell proliferation, differentiation, survival, and death (1). Two recently described cytokines, APRIL and BLyS (also called BAFF, THANK, TALL-1, and zTNF4), form a new subfamily of TNF-like ligands (2–7). BLyS and APRIL are synthesized as type II transmembrane proteins and proteolytically cleaved at a multibasic motif (4, 8, 9). APRIL is processed intracellularly by furin convertase prior to its secretion, thus acting solely as a secreted factor, whereas BLyS is released from the cell surface by processing of membrane-bound BLyS (4, 8, 9). Two members of the TNF receptor family, the transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA), have been identified as receptors that can bind both APRIL and BLyS with comparable efficacy (7, 10–13). More recently, a third receptor was identified that is unique to BLyS (BAFF-R or BR3); this receptor further enhances the complexity of this subfamily (14, 15). Expression of BCMA, TACI, and BR3 is observed on B cells, whereas TACI is also expressed on activated T cells (16).
A clue to the role of this receptor/ligand subfamily in immunity came from the observation that treatment of mice with a soluble decoy form of TACI or BCMA (TACI-Fc or BCMA-Fc) led to reduced B cell numbers and a block in the humoral response (10, 12, 13, 17, 18). These effects have been attributed to BLyS sequestration, as (a) BLyS acts as a costimulator of B cells in the presence of anti-IgM antibodies (3–5); (b) in vivo administration of a soluble form of BLyS disrupts spleen architecture due to increased B cell numbers (3); (c) mice that express BLyS as a transgene have enlarged spleens and lymph nodes and display autoimmunity due to B cell expansion as a result of increased survival of normally deleted B cells (6, 7, 19); and (d) BLyS-deficient mice have a phenotype comparable to that of TACI-Fc– or BCMA-Fc–treated mice, i.e., almost complete loss of mature B cells and a severely decreased humoral response (20, 21). This last observation in particular suggests that BlyS binding to TACI and/or BCMA is essential for B cell survival and function. Paradoxically, TACI-deficient mice show B cell expansion rather than death, whereas BCMA knockout mice have no overt phenotype (20, 22–24). This points to an unexpected role for TACI in negative regulation of B cell growth and a possibly redundant role for BCMA. Despite the B cell expansion, in at least one report the T cell–dependent humoral response appeared normal in the TACI knockout mice (22). A significant decrease is observed, however, in responses to T cell–independent type 2 (TI-2) antigens (22, 23). These apparently contradictory findings in BLyS, TACI, and BCMA knockout mice were at least partly clarified with the identification of BR3, the third receptor for BLyS. This receptor appears to mediate most of the B cell survival signal elicited by BLyS and explains to a large extent the phenotype of the BLyS transgenic (Tg) animals.
In addition to a defect in B cell immunity, BLyS Tg mice have increased numbers of activated CD4+ and CD8+ T cells (7, 19). Although these effects have not been characterized to the same extent as have the changes in B cell homeostasis, they do point to a possible role for BLyS in T cell activation. In agreement, BLyS was recently reported to provide costimulation for suboptimally activated T cells (17).
The role of APRIL in immune regulation is less well defined. APRIL was originally described to stimulate growth of tumor cells in vitro and in vivo (2). Recently, reports described the potential of recombinant APRIL to act as a costimulator of primary B and T cells in vitro and to stimulate IgM production by peripheral blood B cells in vitro (18, 25). Similar to BLyS, in vivo application of APRIL results in splenomegaly due to expansion of the B cell population and an increase in the percentage of activated T cells (18), suggesting a role for APRIL in lymphoid homeostasis as well. To better characterize this function, we generated APRIL Tg mice and have determined the role of APRIL in lymphoid development and activation.
Generation and screening of lck-APRIL Tg mice. The human APRIL cDNA coding region was inserted into a targeting vector containing the lck distal promoter fused to human growth hormone (hGH) intronic and polyadenylation sequences (26). The resulting construct was injected into BALB/c × C57BL/6 embryos, and Tg founders and offspring were screened by PCR of tail DNA using oligonucleotide forward primer H19 5′-ATGGATTACAAAGACGATGACG-3′ and reverse primer H14 5′-TCACAGTTTCACA AACCCCAGG-3′. The APRIL Tg mice were backcrossed on a C57BL/6 background. Mice used in experiments had been backcrossed at least five times onto the C57BL/6 background.
RT-PCR. Naive CD4+ T cells were isolated from the T cell receptor (TCR) Tg DO11.10 mice as described (27). Isolated T cells were restimulated (in the absence of irradiated splenocytes) with 5 mM of the relevant ovalbumin (OVA) peptide and in the presence of IL-12 and anti–IL-4 to generate Th1 cells, or in the presence of IL-4, anti–IL-12, and anti–IFN-γ to generate Th2 cells. Total RNA was isolated from 2 × 106 naive or activated T cells, and cDNA was prepared using a cDNA synthesis kit (Amersham Pharmacia Biotech, Piscataway, New Jersey, USA). cDNA was screened for expression of murine APRIL (700 bp) employing primers H195 (forward: 5′-AGCCTCATCTCCAGGCCACAT-3′) and H194 (reverse: 5′-AAACCCCAGGAATGTTCCATGCG-3′) and murine BLyS (930 bp) employing primers H198 (forward: 5′-ATGGATGAGTCTGCAAAGACCCTG-3′) and H199 (reverse: 5′-TTACAGCAGTTTTAGG GCACCAAAG-3′). As a control, cDNA was screened for GAPDH as described (28). Samples were amplified for 35 cycles (APRIL and BLyS) or 26 cycles (GAPDH).
Total RNA was isolated from splenocytes and reverse-transcribed to cDNA. Aliquots from cDNA preparations were amplified by PCR, generating the complete cDNA of human APRIL (780 bp) employing primers H14 and H19, the complete cDNA of murine APRIL (700 bp) employing primers H195 and H194, or a 445-bp fragment of actin (forward: 5′-ATCAAGATCCTGACCGAGCG-3′; reverse: 5′-TACTTGCGCTCAGGAGGAGC-3′) (29). The following conditions were used: 1 cycle at 94°C for 5 minutes, followed by 3 cycles at 94°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute, followed by another 35 cycles at 94°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes. The products were resolved on a 1% agarose gel. To ensure that the amplified human APRIL signal was not due to contaminating genomic DNA, the entire procedure was performed in the absence of reverse transcriptase. Moreover, cDNA probes were amplified for Thy1 using primers (forward: 5′-CCATCCAGCATGAGTTCAGCC-3′; reverse: 5′-GCATCCAGGATGTGTTCTGA-3′) that amplify a 300-bp fragment of cDNA and a 700-bp fragment of genomic DNA (genomic mouse DNA was used as a positive control).
Western blot analysis. Cell lysates were prepared in NP-40 buffer (1% NP-40, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mM PMSF in 50 mM Tris, pH 7.4). Following quantification of protein content to ensure equal loading of the cell samples, lysates were separated electrophoretically by SDS-PAGE, then transferred to nitrocellulose. Equal loading and transfer was verified in each experiment by Ponceau S staining of the membrane (Sigma-Aldrich). For analysis of mouse sera, 1 μl of each sample was analyzed by Western blot analysis. Immunoblots were probed using 2 μg/ml of affinity-purified anti-APRIL antibody (8) and developed using the ECL system (Amersham Pharmacia Biotech).
Flow cytometric analysis. Cells were stained with appropriate antibodies and analyzed on an EPICS XL flow cytometer (Beckman Coulter, Fullerton California, USA). Freshly prepared or cultured lymphocytes were resuspended in FACS buffer (1% BSA, 0.05% sodium azide in PBS). Peripheral blood leukocytes (PBLs) were isolated by density gradient centrifugation of EDTA-treated mouse blood over Ficoll-Paque (Amersham Pharmacia Biotech). Labeled anti-mouse anti-CD4 (L3T4), -CD8 (Ly-2), -CD45R/B220 (RA3-6B2), -IgD (11-26c.2a), -IgM (R6-60.2), -CD69 (H1.2F3), -CD62L (L-selectin), -CD44 (IM7), -CD45RB (16A), -CD23 (IgE Fc receptor), -CD24 (HSA, 30F1), -CD21, -CD5, -Vβ8 (F23.1), and -Vβ6 (RR4-7) antibodies, intracellular cytokine staining kit, and hamster anti–Bcl-2/control hamster Ig kit were purchased from BD PharMingen (Heidelberg, Germany). All antibodies were used according to the manufacturer's specifications. For determination of dead cells, propidium iodide (PI; 2 μg/ml) was added to cell suspensions.
Antigen-specific antibody production in vivo. For analysis of T cell–dependent humoral responses, groups of 6- to 8-week-old APRIL Tg mice and littermates were immunized intraperitoneally with 107 plaque-forming unit (pfu) per animal of a recombinant form of Western Reserve strain or a modified version of attenuated Ankara-strain vaccinia virus (rMVA), which expresses the entire envelope (env) gene of HIV-1 strain IIIB (MVAenv) under viral transcriptional control (30). Fourteen days after inoculation (priming), blood was collected from the retro-orbital plexus using a heparinized capillary tube, collected in an Eppendorf tube, and incubated 1–2 hours at room temperature and then overnight at 4°C. The samples were centrifuged, and serum was obtained and stored at –80°C. After serum sample collection, mice received a second intraperitoneal immunization with 107 pfu per animal of MVAenv (boost), and another 14 days later, mice were bled again. For analysis of TI-2 humoral responses, mice were immunized intraperitoneally with 100 μl PBS containing 30 μg of 4-hydroxy-nitrophenacetyl–conjugated Ficoll (NP-Ficoll) (Biosearch Technologies). Serum was collected as described above.
Serum Ig and autoantibody analysis. Murine serum Ig's were quantitated by standard ELISA techniques using isotype-specific capture antibodies and specific biotin-conjugated anti-mouse isotype-specific antibodies (BD PharMingen); mouse Ig standards recommended by the manufacturer were used. Dilutions within a linear range of absorbance were chosen for each experiment. To determine antibody titers against vaccinia antigens, β-galactosidase, or 4-hydroxy-nitrophenacetyl (NP), the respective antigen was coated at 1 μg/ml on ELISA plates. Anti-dsDNA autoantibodies were detected by coating ELISA plates with 2.5 μg/ml grade I calf thymus DNA (Sigma-Aldrich). Serum dilutions within the linear range of absorbance were used for quantification. Absorbance was measured at 492 nm on a Labsystems Multiskan Plus plate reader (Thermo Labsystems, Chicago, Illinois, USA).
Cell isolation. T and B cells were purified by negative selection from lymph nodes or spleens using either B220 or Thy1.2-coupled Dynabeads (Dynal Biotech, Oslo, Norway), respectively, according to the manufacturer's instructions. Cell populations isolated by this method were at least 95% positive for B220 (B cells) or CD3 (T cells). In some experiments, CD62L+ cells were sorted using a Beckman Coulter EPICS ALTRA cell sorter.
Proliferation and survival assays. Cells were plated at 5 × 104 cells per well and proliferation quantified by 3H-thymidine uptake (1 μCi/200 μl) during the final 24 hours of culture. T cell proliferation was induced by anti-CD3 (145-2C11; 0.1 μg/ml) alone or in combination with anti-CD28 (37.51; 0.3 μg/ml). Survival of cultured cells was determined by PI uptake. In some experiments, survival was tested in the absence or presence of Mega-APRIL (at indicated concentrations; ALEXIS Biochemicals Corp., Basel, Switzerland). Treatment of Jurkat cells with recombinant APRIL stimulated proliferation in a dose-dependent manner, confirming the activity of the re-agent (not shown). For this, cells were stained with the respective markers and analyzed by flow cytometry after incubation with 2 μg/ml of PI. BCMA-Fc and TACI-Fc were obtained from ALEXIS Biochemicals Corp.
In vivo T cell response. Staphylococcal enterotoxin B (SEB) (100 μg in 100 μl PBS; Sigma-Aldrich) was injected intraperitoneally into mice, and the response of SEB-reactive T cells was determined by analyzing the percentage of Vβ8+ CD4+ and CD8+ T cells in blood at different time points.
APRIL Tg mice. The expression pattern of APRIL on lymphoid cells remains ill-defined. Injection of recombinant APRIL nonetheless results in expansion of the B cell population and T cell activation (18), suggesting that it plays a role in the immune system. To analyze whether APRIL, like BLyS (18), is expressed on T cells, we purified TCR Tg CD4+ T cells from DO11.10 mice. These T cells can be activated in vitro using an appropriate OVA peptide. Moreover, these T cells can be directed toward a Th1 or Th2 phenotype depending on the cytokines present during activation (27). Using this model system, we analyzed APRIL and BLyS expression by RT-PCR. As reported previously, BLyS expression was upregulated during T cell activation (Figure 1a) (4). Similarly, APRIL expression was detected in activated Th1 or Th2 cells, but not in naive cells (Figure 1a), pointing to a role for this molecule in T cell–dependent immunity.
(a) APRIL expression in activated, but not in naive, T cells. Activated T cells have increased APRIL mRNA expression levels. DO11.10 spleen CD4+ T cells were isolated and activated in vitro toward a Th1 or Th2 phenotype. RNA was prepared from naive, Th1, and Th2 cells and used to analyze the expression of APRIL, BLyS, and GAPDH by RT-PCR. Generation and characterization of APRIL Tg mice. (b) Schematic diagram of constructs used for peripheral T cell–specific expression of the human APRIL transgene. (c) mRNA expression of APRIL in T cells of four different founders. RNA was prepared from purified T cells derived from spleen and lymph nodes of four Tg mice and analyzed by RT-PCR for APRIL RNA expression levels. To ensure that the amplified human APRIL signal was not due to contaminating genomic DNA, cDNA probes were amplified for Thy1 using primers that amplify a 300-bp fragment of cDNA and a 700-bp fragment of genomic DNA (genomic mouse DNA was used as control). (d) Protein expression of APRIL in Tg mouse T cells. Cell lysates were prepared from purified T cells derived from spleen and lymph nodes of four Tg mice, a control littermate, and a C57BL/6 mouse and analyzed in Western blot using anti-human APRIL antibodies (8). (e) Secreted APRIL circulates in serum of APRIL Tg mice. Sera (1 μl) of control and Tg mice were resolved under nonreducing conditions and immunoblots developed with anti-human APRIL antibodies. Recombinant (Rec.) APRIL protein (0.1–5 ng) added to control serum was used as standard.
To study this finding in further detail, we generated a Tg mouse strain expressing human APRIL under the control of the lck distal promoter, which directs transgene expression to mature thymocytes and peripheral T lymphocytes (Figure 1b) (26). We confirmed in ELISA assays that binding of recombinant human APRIL to mouse TACI and BCMA is comparable to its binding to the corresponding human receptors (not shown), which concurs with a recent report that human APRIL has the same binding capacities for human and mouse BCMA (31). In four independent Tg mouse lines, we detected RNA expression of the transgene (Figure 1c). Only two of these transgenes displayed detectable levels of the transgene protein in T cells (Figure 1d), but in no other types of cells tested (not shown). Moreover, sera from 8-month-old mice of the Tg 3919 line showed detectable circulating APRIL levels at concentrations of 1–5 μg/ml (Figure 1e). Tg mice were born at the expected mendelian ratio, and histological analysis at 3–12 weeks of age revealed no gross abnormalities in either of the Tg strains. In addition, semiquantitative RT-PCR analysis of RNA derived from APRIL Tg mouse spleens revealed no alterations in BLyS, TACI, or BCMA RNA expression levels (not shown). Line 3919 was used for subsequent experiments, but similar observations were made in line 3923.
T and B cell composition of lymphoid organs. As recombinant APRIL results in increased spleen weight due to B cell accumulation (18), we compared the spleens of 12 Tg mice and their corresponding non-Tg littermates at 6–12 weeks of age. No significant difference was observed in spleen weight (Figure 2a) or total number of spleen cells (Figure 2b), and immunohistological analysis of APRIL Tg mouse spleen architecture showed no apparent abnormalities (not shown). Furthermore, no alteration was observed in spleen weight or total cell number in older Tg mice tested (age 4–9 months) (not shown). We next analyzed the B and T cell composition in Tg mouse secondary lymphoid organs by FACS analysis. No variation was observed in the percentage of B220+ B cells in spleen, mesenteric lymph nodes, or Peyer's patches (Figure 2c). In contrast, a small increase in the percentage of B220+ B cells was observed in peripheral lymph nodes in all Tg mice tested (n = 10) (Figure 2c). The percentages of both CD4+ and CD8+ T cells were proportionally decreased (Figure 2d), resulting in a significant decrease in absolute T cell numbers in Tg mouse peripheral lymph nodes (Figure 2e). Further analysis of T cells revealed a striking increase in CD62L– CD4+ and CD8+ T cells in secondary lymphoid organs, of which only a small percentage showed CD44 upregulation (Figure 3). Whereas a fivefold increase has been observed in CD62L– CD44high effector T cells in BLyS Tg mice (19), APRIL Tg mice revealed an accumulation of CD62L– CD44low T cells, which do not correspond to classically activated T cells. In agreement, there was no reproducible upregulation of CD25 or downregulation of CD45RB in the T cells in the mice analyzed (not shown).
B and T cell homeostasis in APRIL Tg mice. (a) Unaltered spleen weight in APRIL Tg mice. Spleen weight of mice was measured at the age of 6–12 weeks (n = 12). (b) Unaltered splenocyte and peripheral lymph node cell number in APRIL Tg mice. Splenocyte number was determined in 6- to 12-week-old mice (n = 11). Data represent the mean ± SD. (c) Evaluation of B cell percentage (B220+ cells) in spleen, mesenteric lymph nodes (MLN), Peyer's patches (PP), and peripheral lymph nodes (PLN) (n = 10). (d) Percentage of CD4+ and CD8+ T cell populations in peripheral lymph nodes of APRIL Tg mice (n = 10; 6–12 weeks old). (e) Comparison of absolute B and T cell numbers in peripheral lymph nodes (n = 10). Data are the mean ± SM. The statistical significance of the data was determined using ANOVA. P > 0.05 is considered insignificant, P < 0.05 significant, P < 0.01 very significant, and P < 0.0001 extremely significant. Co, control.
Decreased CD62L (L-selectin) expression in splenic T cells is not accompanied by CD44 upregulation. CD62L versus CD44 expression is shown gated on CD4 and CD8 populations. Downregulation of CD62L was seen in T cells of blood and secondary lymphoid organs; it was already apparent in Tg mouse spleen at 2 weeks of age and was maintained throughout their lives (not shown).
Ectopic APRIL expression prolongs T cell survival in vivo and in vitro, correlating with increased Bcl-2 levels. Recombinant APRIL stimulates B and T cell proliferation in vitro (18). In agreement, purified T cells from APRIL Tg mice showed increased thymidine incorporation following a 2-day stimulation with anti-CD3 in combination with anti-CD28, as well as after stimulation with anti-CD3 alone (Figure 4a). The increased in vitro proliferation of Tg T cells correlated with increased IL-2 production in CD8+ T cells after in vitro stimulation (Table 1).
Increased proliferation of T cells and survival of T cells from APRIL Tg mice in vitro. (a) Increased proliferation of Tg T cells after 2 days' culturing with anti-CD3 alone, or anti-CD3 in combination with anti-CD28. (b) T cell response in APRIL Tg mice in vivo: Delayed deletion of superantigen-responsive Vβ8+ CD4+ cells in APRIL Tg mice. PBLs were stained for CD4, CD8, and Vβ8 on the days indicated (n = 4). This is one representative experiment of four performed. ANOVA confirmed that the differences for CD4+ cells at days 7, 10, and 13 are statistically significant (Table 2). *P < 0.05. The Vβ6+ subpopulation of CD4+ and CD8+ cells was unresponsive at all time points analyzed (not shown). (c and d) Survival of purified T cells cultured for 3 days alone, with anti-CD3, or with anti-CD3 plus anti-CD28. Cells were stained for CD4 (c) or CD8 (d) and PI as described in Methods. (e) Purified T cells of C57BL/6 mice were cultured for 2 days without stimulation, alone or in the presence of 5 μg/ml recombinant APRIL, and PI-stained. (f) Purified CD4+ T cells were cultured for 3 days without stimulation, alone or in the presence of 50 μg/ml of TACI-Fc, BMCA-Fc, or control Ig. Similar results were obtained for CD8+ T cells. All data in a and c–f are the mean ± SD of triplicate determinations of a representative experiment of at least three performed. The statistical significance of the data was determined using ANOVA.
Cytokine production of in vitro activated Tg T cells
To determine whether the increased in vitro T cell proliferation observed can also be detected in vivo, we analyzed T cell activation using injection of SEB, which interacts predominantly with Vβ8+ T cells (32). Treatment of mice with SEB leads to systemic activation of all Vβ8+ T cells. SEB injection leads to an initial increase in the percentage of both Vβ8+ CD4+ and Vβ8+ CD8+ T cells. This initial proliferation is followed by a deletion phase, with clear decreases in Vβ8+ T cell numbers, which eventually reach levels lower than those found before SEB treatment. The decrease in Vβ8+ CD4+ cells is due to apoptosis of SEB-responsive cells (32, 33); Figure 4b shows a representative experiment tracking the percentage of Vβ8+ T cells in PBLs from APRIL Tg mice and littermates following SEB administration. Both Vβ8+ CD4+ and Vβ8+ CD8+ T cells of APRIL Tg mice show expansion similar to that of corresponding non-Tg littermates at day 3, indicating that APRIL does not affect the expansion phase of the T cell response in vivo. In contrast, a significantly delayed deletion of Vβ8+ CD4+ T cells was observed in comparison with SEB-reactive cells of littermates; this was especially evident when the percentage of Vβ8+ CD4+ T cells was compared 7 days after SEB injection. Despite the clear delay in deletion of CD4+ T cells, no significant difference was observed in the CD8 response (Table 2). This lack of effect on the CD8+ T cell response was confirmed by analyzing the CD8 response induced after infection with adenovirus. Using this approach, we observed no effect of ectopic APRIL expression in the CD8+ T cell response to adenovirus (not shown).
P values for data in Figure 4b
To analyze this delayed T cell deletion in further detail, we tested whether ectopic APRIL-expressing T cells have an increased survival capacity in vitro. To this end, purified T cells were cultured for 3 days under various conditions, and survival was determined by measuring PI uptake. Both CD4+ and CD8+ T cells isolated from Tg mice showed significantly increased survival compared with those of littermates (Figure 4, c and d). This difference became evident only when T cells received suboptimal or no stimulus. Increased survival of purified T cells also became evident by quantification of live and dead cell gates by forward/side scatter (Table 3). Increased survival of APRIL Tg T cells was not blocked by BCMA-Fc or TACI-Fc (Figure 4f). In addition, the increased survival of nonactivated T cells could not be mimicked by the addition of recombinant APRIL to T cell cultures (Figure 4e). Taken together, this suggests that the presence of APRIL in in vitro cultures is not sufficient to increase survival of unstimulated T cells, but that Tg T cells receive this survival signal in vivo.
Survival of cultured T cells determined by forward scatter and side scatter
The augmented survival of B cells observed in BLyS Tg mice has been associated with increased Bcl-2 expression levels (19). Bcl-2 expression is especially effective when cells are deprived of survival factors (34, 35). Moreover, Bcl-2 has been shown to delay the deletion of SEB-primed CD4+ T cells (33). We therefore determined Bcl-2 expression in the APRIL Tg mice and control littermates by FACS analysis. Ex vivo CD4+ and CD8+ T cells, but not B cells, displayed a twofold increase in Bcl-2 levels in Tg mice compared with non-Tg littermates, suggesting that elevated Bcl-2 levels may be responsible for the prolonged survival of these cells (Figure 5a).
T cells of Tg mice have increased Bcl-2 levels. (a) Increased Bcl-2 expression in CD4+ and CD8+ T cells, but not B cells of APRIL Tg mice. Ex vivo CD4+ and CD8+ T cells and B220+ B cells from peripheral lymph nodes were analyzed for Bcl-2 expression as described in Methods. Staining in cells of control littermates is shown in black (gray shadow for isotype control, no shadow for Bcl-2 staining); blue shows isotype control, and red shows Bcl-2 in cells of Tg's. One representative staining of four is shown. The average increase in Bcl-2 mean fluorescence intensity was 1.5 ± 0.19–fold in Tg CD4 cells (mean ± SD, n = 4), 1.8 ± 0.4–fold in CD8 cells, and 1.1 ± 0.05–fold in B220+ cells. (b) Increased Bcl-2 expression in sorted CD62L+ T cells of Tg mice. Staining in cells of control littermates is shown in black (gray shadow for isotype control, no shadow for Bcl-2 staining); blue shows isotype control, and red shows Bcl-2 in cells of Tg's. One representative staining of three is shown. (c) T cell proliferation of sorted CD62L+ T cells after 2 days' culture with anti-CD3 in combination with anti-CD28. (d) Survival of sorted CD62L+ T cells cultured for 3 days without stimulation. Data in c and d are the mean ± SD of triplicate determinations of a representative experiment of at least three performed. The statistical significance of the data was determined using ANOVA.
The increased proliferation, survival, and Bcl-2 expression may be due to the presence of the relatively large CD62L– T cell fraction in the Tg animals, which might represent activated T cells. To exclude this possibility, we analyzed CD62L+ T cells, which represent the naive T cell fraction. Like unsorted T cell populations, Tg mouse CD62L+ T cells showed increased T cell proliferation following anti-CD3/anti-CD28 activation, as well as prolonged survival in 3-day cultures (Figure 5, c and d). In addition, an increase in Bcl-2 expression was detected on sorted Tg mouse CD62L+ T cells compared with those of littermates (Figure 5b). This indicates that the survival and proliferation effects described are not due to the increase in CD62L– T cells in the Tg mice but are a direct result of APRIL expression on naive T cells.
APRIL Tg mice display normal B cell development in spleen. Only mature B cells can recirculate and enter lymphoid follicles of spleen and lymph nodes. Two spleen B cell populations, termed type 1 (T1) and type 2 (T2) transitional B cells, were identified as precursors for mature B cells (36). BLyS preferentially supports the survival of T2 B cells (37). To analyze whether Tg expression of APRIL had a similar effect on the B cell compartment, we determined the composition in the spleen. T1 and T2 transitional B cells and marginal zone B cells can be distinguished on the basis of IgM, CD21, and CD23 expression (36). The T1 and T2 transitional B cell subpopulations are both IgMbright, whereas only T2 cells are CD21bright CD23+, which also distinguishes them from CD21bright CD23– marginal zone B cells (36). Triple staining of spleen B cells from mice Tg for APRIL showed no significant difference in the percentage of T1, T2, mature, or marginal B cells (Figure 6). T1 B cells are the precursors of T2 and mature B cells (36), and T1 B cells are already detectable in 1-week-old mice, whereas T2 and mature B cells only become clearly detectable in mice at about 3 weeks of age (36). To exclude that APRIL influences the kinetics of B cell development, we followed T1 and T2 B cell formation in mice at various ages; we found that T1 and T2 B cell development is comparable between APRIL Tg mice and littermates (Figure 6). Notably, B cells in peripheral lymph nodes of APRIL Tg mice correspond to mature (IgM+, CD23+, CD21bright, and HSA+) B cells (not shown). The increased constitutive IgM levels in sera of APRIL Tg mice suggested an alteration in the B1 cell population (38). B1 cells are characterized by B220 and IgM expression, but by a lack of CD23 expression; CD5 expression analysis allows a further subdivision into B1a (CD5+) and B1b (CD5–) subpopulations. We thus determined the percentages of these cells in the peritoneum. As shown in Table 4, no significant differences were found upon analysis of APRIL Tg T cells compared with those of littermates.
Ectopic APRIL expression does not influence B cell development in spleen. Splenocytes were analyzed for their content of T1 (IgMbright CD21– CD23–), T2 (IgMbright CD21bright CD23+), mature (IgM+ CD21bright CD23+), and marginal zone (IgMbright CD21bright CD23-) B cells (n = 20).
No altered percentages of peritoneal B cell populations in APRIL Tg's
Ectopic APRIL expression moderately alters the T cell–dependent humoral immune response. APRIL stimulates IgM production by peripheral blood B cells in vitro (25). We therefore compared serum IgM concentrations between APRIL Tg mice and littermates. IgM serum concentrations of Tg mice showed an approximately twofold increase, whereas IgG levels were comparable between Tg mice and littermates (Figure 7, a and b). The twofold difference in IgM serum levels between Tg mice and littermates persisted in older Tg mice (at age 4–9 months; not shown). No increase was found in anti-DNA antibodies in Tg mouse serum (Figure 7c). As ectopic APRIL expression resulted in increased serum IgM levels, we tested whether the humoral response is also modulated in APRIL Tg mice. Mice were challenged with a modified version of Ankara-strain vaccinia virus (MVA) expressing β-galactosidase (gal), a T-dependent antigen (30, 39). Antibody (IgM and IgG) levels against virus and antigen were measured 14 days after virus inoculation. Serum samples of APRIL Tg mice showed increased levels (approximately twofold) of IgM anti-virus antibodies compared with serum samples of control mice, while IgG induction was similar in these mice (Figure 7d). The IgG2a/IgG1 ratio revealed a Th1 response in both Tg and control mice (not shown). A second immunization (boost) with MVAgal virus resulted in a moderate but nonsignificant increase in IgG in the Tg compared with that of non-Tg littermates (Figure 7d). Similar data were obtained for the antibody response to the recombinant antigen (not shown).
Moderate alteration of serum Ig levels and T cell–dependent humoral responses in APRIL Tg mice. Comparison of IgM (a) and IgG (b) levels in APRIL Tg mice (n = 10 in a; n = 6 in b). (c) Anti-DNA autoantibodies were not elevated in APRIL Tg mice. For comparison, pooled sera of five MRL-lpr mice were used at the same dilution (1:100) as those of APRIL Tg mice and littermates, and at a 1:1,600 dilution to obtain a comparable OD value. (d) T cell–dependent humoral response to a modified version of attenuated Ankara-strain vaccinia virus (rMVA) in APRIL Tg mice (n = 4). Serum IgM and IgG levels from 8-week-old mice before immunization (preimmune), 15 days after immunization (priming), and 15 days after second immunization (boost). Statistical significance was determined using ANOVA.
We also analyzed the humoral response in APRIL Tg mice to a wild-type strain of vaccinia virus (a recombinant form of the Western Reserve strain), which showed a pattern similar to that observed for MVA, i.e., a twofold increase in antigen-specific IgM production within 15 days of immunization (not shown).
Elevated TI-2 humoral response in APRIL Tg mice. Polyvalent antigens such as pneumococcal polysaccharides and Ficoll-conjugated antigens are so-called T cell–independent type 2 (TI-2) antigens, as they were originally described to induce T cell–independent immune responses (40). TACI-deficient mice revealed depressed responsiveness to TI-2 antigens, manifesting the importance of that receptor for developing TI-2 responses (22). APRIL is a known TACI ligand; we therefore evaluated the role of APRIL in the generation of the TI-2 humoral response. Immunization of APRIL Tg mice with a model TI-2 antigen, NP-Ficoll, resulted in an increase in IgG response compared with that of non-Tg littermates (Figure 8a). In addition, the IgM response was significantly elevated on day 8 (Figure 8a). The increased IgG response is particularly evident for IgG1, IgG2a, and IgG2b, as they remained significantly elevated even into day 14 after TNP-Ficoll treatment (Figure 8b). IgG3 levels were also significantly increased at day 8, but we observed a leveling at later time points (Figure 8c).
TI-2 humoral response in APRIL Tg mice. Groups (n = 6) of APRIL Tg mice and littermates were immunized with 30 μg NP-Ficoll, a TI-2 antigen, and serum Ig levels were determined by ELISA. (a) IgM and total IgG response to NP-Ficoll. (b) Distribution of IgG1, IgG2a, and IgG2b isotypes. For comparison, IgG2a levels of preimmune sera are shown; similar values were obtained for IgG1 and IgG2b levels in preimmune sera. (c) IgG3 response to NP-Ficoll. The statistical significance of the data was determined using ANOVA.
The TNF-related ligands APRIL and BLyS and their cognate receptors BCMA and TACI form a two-ligand/two-receptor system implicated in B and T cell stimulation. APRIL and BLyS are both expressed in hematopoietic cells, and the functional differences between APRIL and BlyS within this two-ligand/two-receptor system have remained largely undefined to date. Analysis of APRIL Tg mice revealed that APRIL acts as an in vitro survival factor for T cells and affects both the humoral and the T cell response in vivo.
T cell survival and proliferation in vitro. T cells derived from APRIL Tg mice show significantly increased proliferation as measured by thymidine incorporation, which became especially evident after activation with anti-CD3/anti-CD28. This concurs with reports proposing a role for APRIL as an in vitro T cell stimulator (18, 41); we also observed that activated T cells divide more rapidly upon the addition of recombinant APRIL (not shown). The elevated production of IL-2 in in vitro activated Tg T cells probably contributes to their increased proliferation. The proliferative effect may be mediated by TACI, an APRIL receptor, whose cell surface expression is upregulated in activated T cells (42); alternatively, nonactivated T cells may express a new, yet-undefined APRIL receptor. Supporting the existence of an additional APRIL receptor is the fact that the Jurkat human leukemia T cell line, which is highly susceptible to APRIL stimulation, does not contain detectable levels of TACI or BCMA mRNA (42, 43) (M. Hahne, unpublished data). It has also recently been reported that fibroblast and epithelial cell lines express a new, still-unidentified APRIL receptor (44).
In addition to the effect on proliferation, we observed a highly significant increase in in vitro survival of ectopically APRIL-expressing T cells under conditions in which cells are deprived of survival factors and receive no or suboptimal activation signals. T cell survival coincides with a twofold increase in Bcl-2 levels in these cells. This would explain the protection observed against apoptosis induced by growth factor deprivation, as Bcl-2 Tg T cells display comparable survival in such assays (34). Nevertheless, we cannot exclude the implication of other survival-supporting molecules in APRIL Tg T cells. In contrast to the effect of APRIL on proliferation, this survival signal cannot be blocked by the addition of TACI-Fc or BCMA-Fc in vitro. It is therefore likely that the survival signal is already in place prior to isolation of the cells. Indeed, we observed that the addition of recombinant APRIL failed to upregulate Bcl-2 and to protect T cells from dying in vitro (not shown), suggesting either that APRIL-induced survival is an indirect effect of in vivo APRIL expression and not directly mediated by an APRIL receptor on the T cell, or that an APRIL receptor–mediated survival signal synergizes with other factors present in vivo but not in vitro.
T cell homeostasis in APRIL Tg animals. Despite increased survival of APRIL Tg T cells in vitro, we observe no conspicuous difference in total T cell numbers in these mice. This correlates with previous observations that Bcl-2 Tg T cells survive in vitro but do not display perturbed T cell homeostasis (34). Nevertheless, we also detected increased in vitro T cell proliferation, which is not mirrored by an increase in T cell numbers or by a difference in the in vivo growth kinetics of SEB-activated T cells. Whether this reflects a difference in signal requirements between in vitro and in vivo proliferation, similar to that reported for IL-2–induced T cell proliferation (45), is presently unclear.
Two interesting differences observed in the Tg T cell pool are the reduction in T cells in peripheral lymph nodes and an increase in the number of CD62L– T cells, the latter of which was already apparent at 2 weeks of age. These events are likely to be connected, as CD62L participates in lymphocyte recruitment from the blood into lymph nodes (46). It has been reported that CD62L expression is not essential for lymphocyte homing to Peyer's patches, mesenteric lymph nodes, or spleen but is required for homing to peripheral lymph nodes (47), which are the only secondary lymphoid organ that displays a decreased T cell count in the APRIL Tg mice. It is thus possible that the decreased percentage of T cells in peripheral lymph nodes of APRIL Tg mice is the consequence of diminished T cell homing capacity due to their decreased CD62L expression.
We do not observe complete T cell activation in APRIL Tg mice, whereas full T cell activation was previously reported for mice that had received five consecutive injections of recombinant APRIL (18). This is probably due to the levels attained using these different approaches, also reflected by the differential effects on the B cell compartment. Moreover, injection of BLyS into mice or ectopic BLyS expression results in an increased percentage of activated T cells (3, 19). Whereas ectopic BLyS expression results in an increase in CD62L–CD44high effector T cells (19), we observed a marked accumulation of CD62L– CD44low T cells in APRIL Tg mice. We thus conclude that CD62L– T cell accumulation in APRIL Tg mice is not linked to increased Tg T cell activation. Moreover, the increased in vitro survival and proliferation capacity of Tg T cells is not linked to the increased CD62L– T cell numbers, as these features are also detected when sorted CD62L+ Tg T cells are analyzed.
It has been suggested that APRIL is involved in in vitro T cell proliferation, as anti-APRIL antibodies prevent primary activation in vitro (M. Lenardo, personal communication), although this does not exclude a role for BLyS, which was recently shown to provide in vitro costimulation as well (26). Again, these in vitro observations remain to be evaluated in vivo, and the receptor through which these effects are mediated remains unknown, as no defect in T cell–dependent immunity in TACI–/– mice has been reported (22).
Ectopic APRIL expression attenuates the deletion kinetics of SEB-reactive CD4+ T cells. We found that APRIL attenuated deletion of stimulated CD4+ T cells after SEB treatment. Two independently regulated deletion pathways have been described that regulate the deletion phase of SEB-activated CD4+ T cells (48). One mechanism is due to survival factor deprivation, which can be blocked by Bcl-2, whereas the other pathway is mediated by Fas-induced apoptosis (48). The retarded deletion phase indeed reflects previous observations in Bcl-2 Tg mice, confirming the importance of this molecule in the survival of APRIL Tg T cells. In contrast, deletion of SEB-reactive CD8+ T cells is regulated by perforin (49), and this decline is not prevented by constitutive expression of Bcl-2 (50), even though in vitro survival is observed. These data are therefore consistent with APRIL-induced Bcl-2 expression. Similarly, adenovirus-specific CD8+ T cell responses are not altered in APRIL Tg mice.
Differential APRIL and BLyS function in B cell development. Tg animals that overexpress BLyS have increased numbers of mature B cells, resulting in enlarged spleens, Peyer's patches, and lymph nodes (6, 7, 19). We detected no increase in size or weight of secondary lymphoid organs in these mice, nor was B cell accumulation observed in spleen, mesenteric lymph nodes, or Peyer's patches, although an increased percentage of mature B cells was detected in peripheral lymph nodes. This increase was not reflected in absolute cell numbers, however, but was due to a decrease in T cells in the lymph nodes. A possible explanation for this difference may be that the BLyS expression levels in BLyS Tg mice, in which the transgene is under the control of an antitrypsin or actin promoter, are much higher than those in APRIL Tg mice (6, 19). The observation that in vivo administration of relatively large amounts of recombinant APRIL leads to B cell accumulation in the spleen, resulting in increased spleen weight (18), supports this idea. Exposure to large amounts of APRIL protein may not be physiologically relevant, however, and may induce signals in addition to those that we observed with ectopic APRIL expression. An alternative and, in our view, more likely explanation for the difference between APRIL and BLyS Tg mice is that APRIL and BLyS serve different functions within the immune system. This is supported by the fact that the development of most peripheral B cell populations is severely affected in BLyS-deficient mice, indicating that APRIL cannot compensate for the missing B cell survival signals (20, 21). The recent identification of a specific receptor for BLyS (BAFF-R or BR3) on B cells supports this conclusion. Disruption of this receptor in A/WySnJ mice results in a lack of peripheral B cells. This, combined with the surprising observation that TACI-deficient mice actually accumulate B cells (22, 23), whereas deletion of the BCMA gene has no effect on lymphocyte homeostasis (20, 24), indicates that the in vivo survival signals elicited by BLyS are mediated via this novel receptor. Our results support this conclusion, as ectopic expression of APRIL, with its potential to bind to BCMA or TACI but not BR3, does not affect B cell development and homeostasis.
APRIL plays a regulatory role in humoral responses. Although ectopic APRIL expression did not alter either B2 or B1 B cell numbers, we observed a significant (about twofold) increase in serum IgM levels in APRIL Tg mice, while serum IgG levels were unaffected. TACI–/– mice were shown in one report to have the opposite phenotype, i.e., decreased serum IgM but not IgG concentrations (22). This, combined with the observation that APRIL can promote IgM secretion in vitro (18), suggests that APRIL/TACI interactions may have a role in this secretion. Significantly elevated (up to tenfold) serum IgM and IgG concentrations were also found for BLyS Tg mice (6, 7, 19); furthermore, these mice show greatly increased autoantibody production, even at the age of 2–4 months (6, 7, 19). These differences between ectopic APRIL and BLyS expression can be attributed to the capacity of BLyS not only to bind to TACI and promote IgM secretion, but also to bind to BR3 and promote massive expansion of the B cell compartment. This B cell hyperplasia results in increased serum Ig concentrations, autoantibody production, and B cell responses. Under the conditions of our study, APRIL does not promote B cell hyperplasia and thus does not induce these responses.
In addition to an increase in serum IgM levels, we observed an elevation of T cell–dependent humoral responses in APRIL Tg mice. This increase in antiviral B cell responses is only observed for IgM, whereas IgGs are induced similarly in Tg mice and littermates. It thus appears that APRIL promotes B cell secretion of IgM but is incapable of inducing a class switch in a T cell–dependent response, and of promoting IgG secretion.
Unlike the effect on the T cell–dependent humoral response, APRIL Tg mice had a significantly increased IgG response to a TI-2 antigen at all time points tested. An increased IgM response was also seen 7 days after immunization but equalized later after immunization. Marginal zone B cells play an important role in responding to TI-2 antigens (38, 51). Marginal zone macrophages retain TI-2 antigens and are thus suggested to participate in the generation of TI-2 responses (52). As APRIL is expressed in monocyte/macro-phages (53), this raises the possibility that endogenous APRIL secreted by macrophages within the marginal zone mediates a stimulatory signal to B cells during a TI-2 humoral response. APRIL Tg mice displayed, nonetheless, no alterations in marginal zone B cells, as is also the case for TACI–/– mice (22).
TACI–/– mice are reported to have a block in the TI-2 humoral response (22, 23), indicating a crucial role for TACI in this response. TACI is expressed on B cells; based on our observations, we suggest that the APRIL/TACI interaction participates in TI-2 humoral response as well. TACI–/– mice fail to develop IgM or IgG responses after challenge with TI-2 antigens. As ectopic APRIL expression results in elevated IgM and IgG levels during TI-2 responses, it is likely that TACI receives this stimulatory signal from APRIL. Whether BLyS contributes to TACI signaling is not clear. Sequestering BLyS by BR3-Fc injection is reported to affect TI-2 IgG responses, although IgM responses were not examined (15); this suggests a role for BLyS in this response. This role may nonetheless be exerted through BR3, since A/WySnJ mice, which lack functional BR3, develop a normal IgM response to the TI-2 antigen NP-Ficoll, while IgG responses are severely affected (54). Taken together, the data appear to indicate that the combined action of BLyS and APRIL through BR3 and TACI is essential in the maintenance of, and/or class switching in, the response against TI-2 antigens.
In conclusion, our data provide evidence that APRIL signals survival of T cells, and that this affects the deletion phase of a CD4+ T cell response in vivo. In addition to an effect on the T cell response, APRIL mice display an increase in T cell–dependent IgM responses and TI-2 IgM and IgG responses. As BLyS plays a critical role in B cell responses, BLyS-blocking reagents are presently discussed to treat autoimmunity in humans (55). Nevertheless, further experiments are needed to elucidate whether APRIL and BLyS signals are redundant or are elicited under distinct circumstances. In contrast, the effect of APRIL on T cell survival is unique. Whether this survival signal is also crucial for the T cell response is a question that must await the generation of APRIL knockout mice. Our data nonetheless show that APRIL is involved in both T and B cell responses.
We thank Cathy Mark for critical reading of the manuscript, Santiago Rodriguez and Lucio Gomez for their invaluable assistance during animal experiments, the Centro Nacional de Biotecnología for excellent services, and P. Schneider for reagents. M. López-Fraga is supported by the Consejería de Educación y Cultura de la Comunidad de Madrid and financed by the Fondo Social Europeo. F.A. Elustondo received a fellowship from the Gobierno Vasco. J. de Jong is supported by the Dutch Cancer Society and J.V. Stein by the Human Frontiers Science Program. The Department of Immunology and Oncology was founded and is supported by the Spanish Council for Scientific Research and by Pharmacia Corp. C.E. Carvalho-Pinto is supported by the Brazilian Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior Foundation and by the Universidade Federal Fluminense, Niteroi–Rio de Janeiro, Brazil.
Jan Paul Medema and Michael Hahne are co-senior authors.
Copyright © 2015
American Society for Clinical Investigation


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I read about the Little Flower School in T Magazine about a year or so ago. Since then, I have been hoping to take a class with them. This past weekend, I finally got the chance. You can read more about the teachers (Nicolette and Sarah) and their school here, but I will just say that the class was amazing. They provide all the tools and flowers, and it was all set up in a beautiful studio in Red Hook, Brooklyn. I was extremely impressed and I am hoping to take a class with them again soon. Here are some things that I learned about Floral Design.
1. It's really hard to do well, and it is quite humbling.
2. Color: Each flower has nuance, look for all the colors in the flower and it's petals, it will help you pair it with others. Like this flower in the middle in the shot below, it has burgundy, red, gold, and purple in the center. Amazing! So, observe undertones and overtones. They also advised to start with a simple palette, restraint in color is hard, but better.
3. Build a Foundation: One thing they taught us was how to start building an arrangement, and much like a stew, you have to start with a foundation. Since this was a winter citrus class, we started with citrus, eucalyptus, and cedar. Once we had built our base, we could move onto flowers. Typically, your foundation will be heavier items, like citrus for example. Lastly, keep the middle open, this doesn't mean no flowers, but airy.
4. Focal Flower: Choose your focal flower. So, which flower/s are going to be the star? To continue the analogy, what kind of soup is it, what is the main ingredient? Don't use too many ingredients. If you have two similar flowers next to each other, make them different somehow, either one higher than the other, or one more bloomed than the other, etc. And, consider the stems too, some stems are beautiful and beg to be seen, others are not. Lastly, you can also remove leaves along the stem when necessary.
5. Wispy Gesture: These are flowers that are more delicate and added at the end, like a garnish. They sit above the arrangement.
6. Room to Breathe: Make sure that there is air around each flower, and remember that they will continue to bloom, so you need to give them room to grow.
7. Shape: Asymmetry is advised, one side should be higher than the other, and you should think of the arrangement in two halves/two sides. Also, start with an idea of what kind of shape you are trying to achieve.
8. Connection Flowers: Some flowers, as we saw above, have more than one color and can tie parts of the arrangement together. So if one side is pink and the other orange, you can insert a flower in between that has both tones.
8. Size: Generally speaking, the arrangement will be 1.5 times larger than the vase.
9. Tools: Chicken wire is better than foam, and environmentally friendlier too. Also, another great tip, use a lazy Susan so that you can turn the arrangement while you work.
10. Cut the Stems on the Bias: More surface area = more water for the flowers.
11. Style: I think the biggest lesson was have your own style, Nicolette and Sarah have their own style, they don't follow anyone's rules but their own and I think this is why their work stands out. I am inspired by what I learned, and will definitely try to emulate them, but I am also going to experiment, and see what I can come up with. Floral design is an art, and I love it!