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American Real Estate and Urban Economics Association
Real Estate Economics
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1978, Volume 6, Issue 4
1978, Volume 6, Issue 3
- 241-246 The Mythological Foundations of United States Housing and Urban Development Policy
by William G. Grigsby - 247-270 Urban Growth: A View from the Supply Side
by Michael A. Goldberg & Blake Allan - 271-286 The Land Development Corporation: A System for Selling Rural Real Estate
by Hubert B. Stroud - 287-304 The Impact of Market Experience Upon Appraisers' Energy Awareness
by Hans R. Isakson & Richard L. Haney - 305-326 The Impact of Urban Development on Auto Ownership and Transit Use
by John F. Kain & Gary R. Fauth - 327-334 A Cynic's View of Zoning Reform
by Hugh O. Nourse - 335-336 Zoning Reform: Comment
by Daniel R. Mandelker - 337-340 Comment on a Cynic's View of Zoning Reform
by Claude Gruen - 341-342 Response to Gruen and Mandelker
by Hugh O. Nourse
1978, Volume 6, Issue 2
- 129-152 Distributional Consequences of Alternative Mortgage Instruments
by Kerry D. Vandell - 153-163 A Theory of Vacant Urban Land
by Robert E. Schenk - 164-174 Time on the Market and Selling Price
by Norman G. Miller - 175-185 Avoiding Both Disinvestment and Speculation in Private Multifamily Housing
by Rolf Goetz - 186-203 The Gatekeeper Appraiser's Role in an Era of Higher Energy Prices
by Richard L. Haney & Melvin R. Crask & Hans R. Isakson - 204-226 The Implications of Demand Instability for the Behavior of Firms: The Case of Residential Construction
by Charles F. Manski & Kenneth T. Rosen
1978, Volume 6, Issue 1
- 1-21 House Buying Behavior: An Empirical Study in Cross-Cultural Buyer Behavior
by Donald J. Hempel & Subhash C. Jain - 22-36 Real Estate Investment Perception: A Multidimensional Analysis
by Larry E. Wofford & R. Keith Preddy - 37-49 Inflation and Real Estate Investment Value
by Kenneth M. Lusht - 50-69 Multicollinearity and Reduced-Form Price Equations For Residential Markets: An Evaluation of Alternative Estimation Methods
by George W. Gau & Daniel B. Kohlhepp - 70-85 The Correct use of Confidence Intervals and Regression Analysis in Determining the Value of Residential Homes
by Donald R. Epley & William Burns - 86-104 Uses and Abuses of Residential Building Permits in Forecasting Private Housing Starts
by Albert M. Teplin
1977, Volume 5, Issue 4
- 399-410 Real Estate Portfolio Revision
by Richard J. Curcio & James P. Gaines - 411-433 Simulation Analysis of Alternative Mortgage Instruments
by Al Field & Henry J. Cassidy - 434-454 The Changing Spatial Relationship Between Residences and Worksites in the New York Metropolitan Region: Implications for Public Policy
by Pearl Kamer - 455-462 An Application of Hedonic Indexing Methods to Monthly Changes in Housing Prices: 1965-1975
by Michael G. Ferri - 463-472 A Survey and Comparison of Redlining Influences In Urban Mortgage Lending Markets
by Peter M. Hutchinson & James R. Ostas & J. David Reed - 473-481 Exploratory Research on the Redlining Phenomenon
by Roger S. Ahlbrandt - 482-502 Is There A "New" Internal Rate of Return Literature?
by Austin J. Jaffe
1977, Volume 5, Issue 3
- 255-278 What A National Commission on Neighborhoods Could Do
by Robert K. Yin - 279-301 Environmental Impact Evaluation, Land Use Planning, and the Housing Consumer
by Franklin J. James & Thomas Muller - 302-312 Residential Mortgage Markets: Current Developments and Future Prospects
by Donald M. Kaplan & B.G. Hartzog - 313-336 Impact of New Housing Construction on Racial Patterns
by Ernest Erber - 337-354 Housing Statistics: Some Sources and Some Limitations
by Phillip E. Kidd - 355-360 The Controversy Over Homeownership Affordability
by Bernard J. Frieden & Arthur P. Solomon - 366-379 The Housing Crisis and the Homeownership Incentive Plan
by Kenneth T. Rosen - 386-387 Response to Burnham and Robinson
by Kenneth T. Rosen
1977, Volume 5, Issue 2
- 141-146 Housing the Nation-Part I
by Robert W. Lake - 147-170 A National Policy and Budgetary Framework for Housing and Community Development
by Arthur P. Solomon - 171-188 Housing Policy in a Nongrowth Era: Problems of the Northeast United States
by Frank S. Kristof - 189-208 The Effect of Regional Growth Characteristics on Regional Housing Prices
by Clayton P. Pritchett - 209-226 The Affordability of New Homes
by John C. Weicher - 227-241 The Neighborhood Effects of Filtering
by Michael A. Stegman - 242-253 The Uncharted Trend: Toward Increased Public-Private Cooperation in Housing Development
by Donald E. Priest
1977, Volume 5, Issue 1
- 1-43 Homeownership Effects of Alternative Mortgage Instruments
by James Follain & Raymond Struyk - 44-67 Regional Market Variations: The Northeast Versus The South
by George Sternlieb & James W. Hughes - 68-84 Public Policies for the Preservation of Capital in Older Areas
by Roger S. Ahlbrandt - 85-110 Taxation of Residential Property in New York City: The Sources of Differential Treatment
by Matthew P. Drennan & Emanuel Tobier - 111-127 The Suburbanization of Industry and Its Effects Upon Labor Force Participation Rates of Suburban Women
by Marilyn Rubin - 128-138 A Vacancy Transfer Model of the Structure of the Local Housing Market
by Gary Sands
1976, Volume 4, Issue 3
- 7-18 Housing Production, Consumption, Statistics, and Policy
by Robert Moore Fisher - 19-47 Toward a Model of Real Estate Project Demand Under Uncertainty: An Exploratory Example of Market Simulation
by Wayne R. Archer - 49-67 FMRR and Duration: Implications for Real Estate Investment Analysis
by Guilford C. Babcock & M. Chapman Findlay & Stephen D. Messner - 69-83 Reinvestment Rates and the Sensitivity of Rates of Return in Real Estate Investment
by George W. Gau & Daniel B. Kohlhepp - 85-92 Local Mechanisms for Locational Efficiency
by John P. Blair - 93-108 "Overcrowded" Households in the United States
by Ernest M. Fisher
1976, Volume 4, Issue 2
- 7-18 Narrow Versus Wide Stratification of Data in the Development of Regression Appraisal Models
by T. Gregory Morton - 19-31 An Econometric Analysis of Urban Travel Behavior Between Residence and Work Site
by C. F. Sirmans - 33-55 Controlling for Uncertainty Through Computer Applications of PERT/CPM to Real Estate Project Analysis
by Nicholas Ordway - 57-75 An Empirical Study of Time on Market Using Multidimensional Segmentation of Housing Markets
by Jacob Belkin & Donald J. Hempel & Dennis W. McLeavey - 77-89 A Method for Building a Property Values Index for use in Local Public Policy Decisions
by Warren Fisher & Norman Starler & Ann Fisher - 91-99 A Comparison of Consumer and Realtor Attitudes Regarding Consumer Issues Affecting the Housing Industry
by Donald W. Hackett & John R. Darling
1976, Volume 4, Issue 1
- 7-21 A Component Analysis of Rates of Return in Real Estate Investment
by George W. Hettenhouse & John J. Dee - 23-39 Forecasting Long-Run Land use Patterns with an Aggregative Model of Urban Housing and Transportation: The Case of Los Angeles
by Dennis R. Capozza - 41-55 The Preferences of Financial Institutions for Construction and Permanent Mortgage Lending on Multi-Unit Residential and Commercial Income Properties
by John M. Mason & Frederick H. Leaffer - 57-81 Rent Control: The Potential for Equity
by Monica R. Lett - 83-95 Ohio Usury Ceilings and Residential Real Estate Development
by Douglas V. Austin & David A. Lindsley - 97-104 Can We Design the Housing Allowance for Learning?
by Hugh O. Nourse
1975, Volume 3, Issue 3
- 7-21 Canada's Federal Mortgage Exchange Corporation
by J. V. Poapst - 23-46 Market Implications of a Direct Cash Assistance Program in Southern Nonmetropolitan Cities
by Michael A. Stegman & Howard J. Sumka - 47-60 Rent Control within the Rental Housing Parameters of 1975
by Frank S. Kristof - 61-80 Condominium Conversion Profiles: Governmental Policy
by George Sternlieb & James W. Hughes - 81-93 Economic and Demographic Factors Affecting Housing Demand into the 1980s and 1990s
by Thomas C. Marcin & John Kokus - 95-111 Variable Rate Mortgage Lending: Some Empirical Results
by James A. Millar & Stanley R. Stansell
1975, Volume 3, Issue 1
- 7-16 Concentration of Rental Housing Property and Rental Housing Markets in Urban Areas
by R. Cherry & E. J. Ford - 17-31 An Econometric Model for Appraising
by Albert M. Church - 33-47 Housing Needs of the Urban Poor: The Problems and Alternatives
by James R. Cooper - 49-72 Life Insurance Companies and the Commercial Mortgage Market, 1960-73
by Thomas R. Robinson - 73-88 A Discriminant Function Analysis of Residential Mortgage Delinquency and Foreclosure
by T. Gregory Morton
1974, Volume 2, Issue 2
- 7-23 Neighborhood Dynamics and Government Policy
by George Sternlieb & James W. Hughes - 25-45 Evaluating the Usefulness of Land use Models for Policy Analysis
by Janet Rothenberg Pack - 47-58 The uses of Urban Research: A Perspective on the Urban Observatory Experience
by William R. Barnes - 59-80 Translating Housing Analysis into Housing Policy
by Sammis B. White - 81-99 Nonmetropolitan and Inner-City Housing Investment Markets
by Michael A. Stegman & Howard J. Sumka - 101-112 Default Risk in Urban Mortgages: A Pittsburgh Prototype Analysis
by Alex O. Williams & William Beranek & James Kenkel
1974, Volume 2, Issue 1
- 5-19 Estimation of Delinquency Risk for Home Mortgage Portfolios
by George M. Furstenberg & R. Jeffrey Green - 21-36 Variable Rate Mortgages: Their use and Impact in the Mortgage and Capital Markets
by Richard G. Marcis - 39-55 School Finance Reform and Property Tax Relief Programs: Their Effect on Urban Fiscal Disparities
by John Callahan & William H. Wilken - 57-73 The Issue of Fractional Assessment
by D. R. Epley - 75-88 An Alternative Approach to Asset Selection Under Uncertainty
by Allen A. Abrahamson & John T. Emery - 89-106 Growth and Prospects of Private Mortgage Insurance Activity
by Chester Rapkin
1973, Volume 1, Issue 2
- 5-20 Evaluation of National Housing Goals
by Ernest M. Fisher - 21-34 Evaluating the Administrative Delivery of Housing Goals
by Charles G. Field - 35-51 Evaluating National Housing Criteria in Canada
by Lawrence B. Smith - 53-72 The Role of State Housing Finance and Development Agencies in Future Federal Housing Programs
by Frank S. Kristof - 73-87 Evaluating the State's Role in Water Pollution Control
by Harvey Lieber & Bruce Rosinoff - 88-103 How the General Accounting Office Evaluates Urban Housing Policies: An Analysis of Criteria and Procedures
by Charles J. Stokes - 104-118 Evaluation of Multiple Subsidy Programs in a Local Market
by Ronald L. Racster & Robert Howard - 119-140 Housing Subsidies and Housing Starts
by Craig Swan - 141-151 Evaluation of Metropolitan Conflict and Cooperation
by John J. Callahan
1973, Volume 1, Issue 1
- 4-7 Real Estate and Urban Land Economics: Illusions of Progress?
by William G. Grigsby - 8-30 Effects of Monetary Policy and Credit Conditions on the Housing Sector
by Steven A. Y. Lin - 31-47 A Locational Analysis of Manufacturing Plants within the City of Chicago: 1959-1968
by Harry D. Hartnett - 48-78 A Computer Simulation Model to Measure the Risk in Real Estate Investment
by Stephen A. Pyhrr - 79-106 A Simple Model of Housing Production and the Abandonment Problem
by Gregory K. Ingram & John F. Kain - 107-122 Growing Scale of Real Estate Firms and Projects
by Leo Grebler - 123-139 Speculative Development of Residential Land and Residential Structures During the 1920's and the 1960's and 1970's
by Ernest M. Fisher - 140-157 New Techniques of Financing Real Estate
by Paul F. Wendt


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Mechanisms imposing the VBeta bias of V14a natural killer T cells and consequences for microbial glycolipid recognition Mouse and human natural killer T (NKT) cells recognize a restricted set of glycosphingolipids presented by CD1d molecules, including self iGb3 and microbial alpha-glycuronosylceramides. The importance of the canonical Valpha 14-Jalpha18 TCR alphachain for antigen recognition by NKT cells is well recognized, but the mechanisms underlying the Vbeta8, Vbeta7, and Vbeta2 bias in mouse have not been explored. To study the influences of thymic selection and the constraints of pairing with Valpha 14-Jalpha 18, we have created a population of mature T cells expressing Valpha 14-Jalpha 18 TCRalpha chain in CD1d-deficient mice and studied its recognition properties in vitro and in vivo. Transgenic cells expressed a diverse Vbeta repertoire but their recognition of endogenous ligands and synthetic iGb3 was restricted to the same biased Vbeta repertoire as expressed in natural NKT cells. In contrast, alpha-GalCer, a synthetic homologue of microbial alpha-glycuronosylceramides, was recognized by a broader set of Vbeta chains, including the biased NKT set but also Vbeta6, Vbeta9, Vbeta10, and Vbeta14. These surprising findings demonstrate that, whereas Vbeta8, Vbeta7, and Vbeta2 represent the optimal solution for recognition of endogenous ligand, many Vbeta chains that are potentially useful for the recognition of foreign lipids fail to be selected in the NKT cell repertoire. The Journal of Experimental Medicine ARTICLE JEM The Rockefeller University Press $8.00 Vol. 203, No. 5, May 15, 2006 1197 1207 www.jem.org/cgi/doi/10.1084/jem.20060418 1197 Mouse V 14-J 18 and human V 24-J 18 NKT cells are innate-like, CD1d-restricted, autoreactive lymphocytes that regulate several infectious, autoimmune, and cancerous conditions (1). Their TCR structure combines a canonical TCR chain with TCR chains that belong to a restricted set of V families including V 8, V 7, V 2 in mice and V 11 in humans (2). Unlike the invariant TCR chain, these TCR chains exhibit a wide diversity of CDR3 junctions (3 5), nearly all of which allow for the recognition of the self-glycosphingolipid iGb3 (6), the microbial cell wall -glycuronosylceramides (7, 8), and their mimic -GalCer. In addition, a fraction of NKT cells was reported to recognize mycobacterial phosphatidylinositolmannosides (9). The mechanisms leading to the striking V bias during development and the consequences on microbial ligand recognition have remained unexplained. For example, it is unclear whether the V bias refl ects pairing preferences with the invariant TCR chain or the predominant infl uence of positive selection or negative selection during thymic development. There are reports of pairing defects of the invariant TCR chain with a V 11 chain in vitro (10) and with V chains lacking the solvent-exposed C FG loop in vitro and in vivo (11), suggesting that many V 14-J 18/V dimers are unstable. In addition, diff erent contributions of individual V 8, V 7, and V 2 families to the affi nity of recognition of -GalCer have been shown (12), suggesting that the V bias may also refl ect thymic selection by lipid ligands. Indeed, recent studies suggested V -specifi c diff erences in avidity for CD1d/endogenous ligand, namely a higher avidity of V 7 chains (13, 14). However, because these studies did not examine the recognition properties of the nonbiased V repertoire before selection by CD1d/ligands, the forces shaping the V bias during thymic development have not been explored. Here, we have created a population of mature V 14-J 18 T cells expressing a diverse, nonbiased V repertoire by expressing a V 14-J 18 Mechanisms imposing the V bias of V 14 natural killer T cells and consequences for microbial glycolipid recognition Datsen G. Wei,1,3 Shane A. Curran,1 Paul B. Savage,4 Luc Teyton,5 and Albert Bendelac1,2 1Committee on Immunology and 2Howard Hughes Medical Institute, University of Chicago, Chicago, IL 60637 3Department of Molecular Biology, Princeton University, Princeton, NJ 08544 4Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 5Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037 Mouse and human natural killer T (NKT) cells recognize a restricted set of glycosphingolipids presented by CD1d molecules, including self iGb3 and microbial -glycuronosylcera mides. The importance of the canonical V 14-J 18 TCR chain for antigen recognition by NKT cells is well recognized, but the mechanisms underlying the V 8, V 7, and V 2 bias in mouse have not been explored. To study the infl uences of thymic selection and the constraints of pairing with V 14-J 18, we have created a population of mature T cells expressing V 14-J 18 TCR chain in CD1d-defi cient mice and studied its recognition properties in vitro and in vivo. Transgenic cells expressed a diverse V repertoire but their recognition of endogenous ligands and synthetic iGb3 was restricted to the same biased V repertoire as expressed in natural NKT cells. In contrast, -GalCer, a synthetic homologue of microbial -glycuronosylceramides, was recognized by a broader set of V chains, including the biased NKT set but also V 6, V 9, V 10, and V 14. These surprising fi ndings demonstrate that, whereas V 8, V 7, and V 2 represent the optimal solution for recognition of endogenous ligand, many V chains that are potentially useful for the recognition of foreign lipids fail to be selected in the NKT cell repertoire. CORRESPONDENCE Albert Bendelac: email@example.com Abbreviations used: DN, double negative; DP, double positive; NKT, natural killer T; Tg, transgenic. Downloaded from jem.rupress.org on May 8, 2009 Published May 1, 2006 1198 V CONTRIBUTION TO GLYCOLIPID RECOGNITION BY NKT CELLS | Wei et al. TCR transgene in CD1d-defi cient (CD1d / ) mice. Our studies demonstrate that the invariant TCR chain could pair indiscriminately with the entire V repertoire, ruling out pairing biases as a substantial factor. Second, we found that only V 7, V 8, and V 2 allowed for recognition of endogenous ligand and iGb3, with a V 7>V 8>V 2 hierarchy of responses paralleling the corresponding V enrichment by thymic selection in vivo, demonstrating a predominant role of positive selection over negative selection in shaping the V repertoire. In contrast, many V families that are not represented in the natural NKT cell repertoire were nevertheless capable of recognition of foreign and microbial lipids, revealing a surprisingly ineffi cient selection of potentially useful TCRs. RESULTS V 14-J 18 transgenic (Tg) cells in CD1d / mice V 14-J 18 TCR chain Tg mice have an increased population of canonical CD44highNK1.1+ NKT cells with the expected V 8, V 7, V 2 bias and CD4 or double negative (DN) phenotype. We previously reported that some Tg lines also harbored in their thymus and spleen a peculiar subset of mature T cells with a CD44low/intNK1.1 DN phenotype and a conspicuous absence of V bias (15). By crossing the V 14-J 18 transgene onto a CD1d / background, we found that the canonical NK1.1+ NKT cells had disappeared, as expected, whereas the CD44low/intNK1.1 DN T cells persisted (Fig. 1, top and middle). Most of the CD44low/int NK1.1 DN T cells lacked endogenous V chains because only 2% were stained by a combination of antibodies against V 2, V 3.2, V 8, and V 11, which otherwise accounted for 26% of the TCR chains expressed by other T cells (Fig. 1, bottom). Thus, it is likely that the DN T cells express the invariant V 14-J 18 TCR chain encoded by the transgene, whereas other T cells express endogenous V s. Consistent with the expression of V 14-J 18, up to 25% of the DN T cells were brightly stained by the -GalCer-CD1d tetramers (Fig. 1, bottom). Such a peculiar DN lineage maturing in the absence of thymic ligand has been described previously in many TCR Tg systems and is believed to be induced by the early expression of the Tg TCR chain at the DN3 stage (as we observed in this Tg line; unpublished data) competing with the pre-TCR chain for signaling (16 20). Importantly, these Tg cells were reported to respond to their ligand in a dosage-dependent manner despite the absence of thymic selection (21). Nonbiased V repertoire of V 14-J 18 Tg DN cells in CD1d / mice We next studied the V repertoire associated with the invariant TCR chain in the V 14-J 18 Tg DN T cells of CD1d / mice. For comparison with another nonselected V repertoire, we used CD4+CD8+ double positive (DP) thymocytes from 2m / .I-Ab / MHC-null mice that express high levels of surface TCR readily stained by anti-V antibodies (22). For comparison with the CD1d-restricted repertoire, we used CD3+NK1.1+ thymocytes from WT B6 mice thymuses, which contain >95% canonical tetramer+ NKT cells (23). Fig. 2 shows that, in dramatic contrast with the biased V 8, V 7, V 2 repertoire of natural NKT cells in WT mice, the CD1d / Tg DN cells expressed a very diverse repertoire. This repertoire appeared relatively close to the nonselected DP thymocytes in MHC-null mice, although signifi cant diff erences were found for some V s suggesting that, in the absence of selection by CD1d, V 14-J 18 imparts only modest pairing bias compared with the average V Figure 1. Mature DN T cells in V 14tg.CD1d / mice. (top) The spleen of V 14tg mice harbors a sizeable population of CD4 CD8 CD3 + DN T cells (boxed in gate b) that is independent of CD1d expression. (middle) Gated DN T cells have a uniform CD44low/intNK1.1 phenotype in V 14tg.CD1d / mice. (bottom) The DN T cells of V 14tg. CD1d / mice do not express endogenous TCR chains; 25% of them are -GalCer-CD1d tetramer+. Note that, in contrast, the CD4 and CD8 T cells (boxed in gate a) in these mice express endogenous V s but are -GalCer- CD1d tetramer-negative. Data are representative of four independent experiments with two mice per group. Downloaded from jem.rupress.org on May 8, 2009 Published May 1, 2006 JEM VOL. 203, May 15, 2006 1199 ARTICLE chain. To further evaluate the diversity of the V repertoire associated with V 14-J 18, we cloned and sequenced multiple CDR3 segments generated by RT-PCR from sorted, CD1d / Tg DN cells. We chose three TCR chains, V 8.2, V 8.1, and V 12, as representatives of V s highly, poorly, or nonenriched in natural NKT lineage cells, respectively. Fig. 3 shows a broad distribution of CDR3 lengths and a broad usage of J segments for each of these TCR chains, with a similar overrepresentation of the J 2 over the J 1 cluster as previously reported for conventional T cells (24, 25). We found 77 94% of unique sequences in each set of TCR chains analyzed, similar to previous fi ndings reported for other T cell populations (26, 27). Together, these results establish the broad diversity of the V repertoire expressed by the CD1d / Tg DN cells. Thus, pairing of V 14-J 18 with TCR chains is largely promiscuous and unlikely to be a signifi cant factor in the V repertoire bias of V 14 NKT cells. Furthermore, comparison of the nonselected V repertoire of CD1d / Tg DN cells with the natural V repertoire of mature NKT thymocytes allowed a precise calculation of the degree of enrichment associated with thymic selection for each V family. Thus, V 2 was enriched 2.6-fold, V 8.2 was enriched 8-fold, and V 7 was enriched 12-fold. A similar percentage of V 7 and V 8.1+8.2 as in the CD1d / Tg DN cells was also found in DP thymocytes obtained from another CD1d / V 14-J 18 TCR Tg strain where the transgene was driven by a CD4 promoter to avoid premature expression at the DN3 stage (unpublished data). Thus, this striking V 7>V 8.2>V 2 hierarchy contrasts with previous estimates derived merely from the relative frequency of V s expressed by NKT cells, where V 8.2>V 7 because it was six times more frequent than V 7. It also diff ers signifi cantly from estimates derived by comparing the NKT lineage repertoire with mainstream DP thymocytes where V 7 enrichment was similar to that of V 8.2 (14). V -specifi c binding of -GalCer-CD1d tetramers -GalCer-CD1d tetramers stain nearly all mouse NKT cells expressing V 14-J 18 combined with V 8, V 7, V 2 (23, 28). In the nonselected V 14-J 18 Tg DN T cells, 69 85% of cells expressing V 8, V 7, and V 2 were tetramer+ (Fig. 4), indicating that these V s are usually, though not always, compatible with -GalCer recognition independently of the CDR3 sequence. Surprisingly, however, 36 84% of cells expressing additional V families that are not signifi cantly represented in the natural NKT cell repertoire (Fig. 2), including V 3, 6, 9, 10, and 14, were also stained by the tetramers. Figure 2. V repertoire of V 14tg.CD1d / DN T cells. The V repertoires of splenic CD3 + DN T cells in V 14tg.CD1d / mice, DP thymocytes in MHC-null mice, and CD3 +NK1.1+ thymic NKT cells in C57BL/6 mice are compared. Histograms show staining by a panel of anti- V mAbs with percentages as indicated. Data are means SD of three independent experiments with at least two mice per group. Downloaded from jem.rupress.org on May 8, 2009 Published May 1, 2006 1200 V CONTRIBUTION TO GLYCOLIPID RECOGNITION BY NKT CELLS | Wei et al. Other V s, such as V 4, 5, 11, 12, and 13 were mostly negative, although they consistently included 3 10% positive cells. Thus, these results reveal many new V families that allow for -GalCer recognition but had escaped prior recognition because of their absence from the natural NKT cell repertoire. As expected, an extended V repertoire was also found for the microbial lipid -glucuronylceramide of which -GalCer is a mimic (unpublished data). V -specifi c induction of CD69 by CD1d/lipid ligands Because iGb3-CD1d tetramers could not stain NKT cells (6), we devised T cell stimulation assays to compare the V repertoires specifi c for iGb3 and -GalCer. Thymocytes have very poor endocytic properties and are therefore defective in the presentation of exogenous lipid ligands requiring endosomal access for processing or loading onto CD1 molecules (reference 29 and unpublished data). Therefore, we used DCs pulsed with antigen to stimulate DN T cells obtained from the spleen of V 14tg.CD1d / mice. After 4 6 h, DN T cells were harvested and stained for CD69. Fig. 5 shows that a large fraction of V 2, 7, and 8 cells up-regulated CD69 in response to DCs pulsed with either iGb3 (20 46%) or - GalCer (56 77%) when compared with unpulsed DCs from CD1d / mice (3 4%). Consistent with the tetramer staining experiments, many additional V s were stimulated by - GalCer, including V 3, 6, 9, 10, 14 (23 63%), whereas others (V 4, 5, 11, 12, 13) gave very weak responses (4 12%). Interestingly, when compared with CD1d / DCs, unpulsed WT DCs weakly but consistently stimulated V 2, 7, and 8 cells (14 33%), suggesting that natural ligand expression was suffi cient to induce CD69 on a subset of cells expressing the V families naturally associated with NKT cells. Thus, in contrast with -GalCer, both the natural ligands expressed by DCs and exogenously added synthetic iGb3 elicited the Figure 3. V 14tg.CD1d / DN T cells have diverse CDR3 sequences. V 8.1- (n = 35), V 8.2- (n = 58), and V 12-CDR3 (n = 96) sequences amplifi ed by RT-PCR from sorted V 14tg.CD1d / DN T cells were cloned and sequenced. (A) Bar graphs of CDR3 lengths (a.a., amino acid). (B) Frequencies of J usage expressed as percentage of total sequences. Note that a similar bias in J 2 cluster usage has been reported for conventional T cells. Copyright Number of unique and duplicate sequences among V 8.1, V 8.2, and V 12. Figure 4. V -specifi c binding of -GalCer-CD1d tetramers to V 14tg.CD1d / DN T cells. Freshly isolated splenocytes from V 14tg.CD1d / mice were stained with -GalCer-CD1d tetramer, CD3 , CD4 and CD8 , and a panel of anti-V mAbs. Dot plots are gated on CD3 +CD4 CD8 cells. Numbers in the top right quadrants indicate the percentages of tetramer+ cells among the V positive cells. Data are mean SD from three individual mice analyzed in three independent experiments. Downloaded from jem.rupress.org on May 8, 2009 Published May 1, 2006 JEM VOL. 203, May 15, 2006 1201 ARTICLE very same V repertoire as expressed by natural NKT cells. Importantly, neither synthetic iGb3 nor naturally expressed ligands stimulated other V s, suggesting that the V bias of natural NKT cells must refl ect positive selection of these V s rather than negative selection of other V s. V -specifi c induction of cell division by CD1d/lipid ligands Although CD69 can be induced by partial TCR signaling, T cell proliferation requires stronger agonist signals (30). Indeed, iGb3 was reported to be an agonist ligand for mouse and human NKT cells and a prime candidate for mediating the positive selection and the expansion of NKT cell precursors in the thymus (6). Therefore, to model the expansion of selective V s during NKT development, we used CFSElabeled DN T cells to compare the mitogenic properties of endogenous and exogenous NKT ligands in a cell culture system. IL-7 was added to prevent the death of unstimulated cells over the 4-d culture period with antigen-pulsed DCs. When DN T cells were incubated with CD1d / DCs alone, a background CFSE dilution of 50%, likely the result of IL-7, was observed for all V s in diff erent experiments. Remarkably, CD1d-expressing WT DCs induced a signifi - cant and selective expansion of V 7 cells (84%) in the absence of added ligands, above the background CFSE dilution observed in V 6 and V 12 cells (45 48%) (Fig. 6). V 2 (57%) and V 8 (62%) were slightly higher than V 6 and V 12. These data suggest that naturally expressed ligands can act as true albeit weak agonists capable of inducing cell proliferation, with the highest affi nity for V 7, similar to the V 7>V 8>V 2 hierarchy observed in the CD69 induction Figure 5. V -specifi c expression of CD69 by antigenstimulated V 14tg.CD1d / DN T cells. Enriched DN T cells from V 14tg.CD1d / spleens were cocultured with CD1d / DCs or WT DCs pulsed with 100 ng/ml iGb3, 1 ng/ml -GalCer, or no antigen. T cells were stimulated for 4 h with -GalCer or for 6 h with iGb3. Percentages of CD69-positive DN T cells gated according to V usage are indicated in top right quadrants as mean SD from four independent experiments. Downloaded from jem.rupress.org on May 8, 2009 Published May 1, 2006 1202 V CONTRIBUTION TO GLYCOLIPID RECOGNITION BY NKT CELLS | Wei et al. assay (compare with Fig. 5). When a low dose of anti-CD3 antibody was used to induce polyclonal activation, all V s underwent similar expansion, demonstrating that the selective V response to naturally expressed NKT ligands was specifi c to these antigens. DCs pulsed with synthetic iGb3 induced the selective expansion of V 2, 7, and 8 cells (89 96%) well above the levels induced by naturally expressed ligands but with a similar V 7>V 8>V 2 hierarchy. In contrast, consistent with the experiments using tetramer staining (Fig. 4) and short-term induction of CD69 (Fig. 5), -GalCer induced a strong expansion not only of V 2, 7, and 8 cells (98 99%) but also V 6 cells (95%), whereas V 12 cells remained close to background, as expected from their lack of CD69 induction. V -specifi c hierarchy in responses to glycolipid ligands To extend the aforementioned fi ndings to the larger V repertoire and to more rigorously quantitate the apparent diff erences in individual V responses, DCs were pulsed with a range of concentrations of -GalCer and iGb3 before coculture with CFSE-labeled DN T cells. Fig. 7 shows the CFSE dilution profi les according to V expression, with statistics displayed whenever dilution was >50% background level. Frequencies of CFSE-negative (>7 divisions) and CFSE-low cells were displayed separately to provide a quantitation of the rate of proliferation in addition to the frequency of cells having proliferated. Furthermore, absolute cell recoveries according to V expression were also recorded (Table I). -GalCer expanded a broad range of V , including V 2, 6, 7, 8.2, 9, 10, and 14. These results closely resembled the patterns found with tetramer staining (Fig. 4) and CD69 induction (Fig. 5). However, one notable exception was V 3, which stained with tetramers (36%) and induced CD69 (23%), yet failed to induce detectable expansion. Closer examination of Figs. 4 and 5 indicated that the fl uorescence intensity of staining with tetramers or CD69 was clearly lower for V 3 than for other responder V s, suggesting lower affi nity as an explanation for the lack of proliferation. V 7 and V 8.2 cells had comparable responses over a range of -GalCer concentrations, as judged by the percentage of cells that diluted CFSE to low or negative levels and by the cellular expansion measured in absolute cell numbers (Table I). Slight diff erences between V 7 and V 8.1+8.2 cells were the result of the heterogeneity of V 8 because V 8.1 was much less responsive than V 8.2 to -GalCer (Fig. 6 and Table I). In a previous report (12), staining of V 8.2 NKT cells with a dimeric CD1d- GalCer ligand (CD1d-IgG1) was somewhat brighter than that of V 7. This apparent discrepancy with our results may refl ect diff erences between the pre- and postselection NKT cell repertoire or, alternatively, indicate that the affi nity estimated by ligand staining does not always correlate well with various forms of functional stimulation. In contrast with V 8.2 and V 7, V 2 cells were 10 times less responsive to -GalCer than either V 7 or V 8 cells, as judged by the 10 times greater amount of -GalCer required for V 2 cells to achieve similar CFSE dilutions as V 8.2 and V 7 cells. The other responder V chains, including V 6, 9, 10, and 14, required concentrations of - GalCer in the ng/ml range for cell expansion, somewhat higher than V 2, though still in the low range compared with peptide antigens of conventional MHC-restricted T cells. Stimulation by iGb3 demonstrated remarkably distinct features (Fig. 7). In general, iGb3 exerted its stimulatory eff ects at concentrations 30 50 times greater than those needed for -GalCer, but it is unclear at present whether this diff erence in potency refl ects a lower affi nity (weak agonist) or diff erent requirement for uptake and presentation by DCs. In contrast with -GalCer and as noted in the CD69 induction assay in Fig. 5, iGb3 nearly exclusively stimulated V 2, V 7, and V 8, Figure 6. V -specifi c proliferation of antigen-stimulated V 14tg. CD1d / DN T cells. CFSE-labeled V 14tg.CD1d / DN T cells were cocultured with antigen-pulsed DCs in the presence of 10 ng/ml IL-7 for 4 d and CFSE dilution profi les were gated according to V expression. DCs were pulsed with no antigen, iGb3 at 50 ng/ml or -GalCer at 10 ng/ml. Control stimulation was with a low dose of 50 ng/ml of anti-CD3 directly added to the culture wells. Data are representative of three independent experiments. Table I. V -specifi c expansion of V 14tg.CD1d / DN T cells stimulated by lipid antigens -GalCer (ng/ml) iGb3 (ng/ml) 0.01 0.1 1 10 0.5 5 50 500 V 2 0.6 0.9 4.2 9.2 0.5 0.7 3.7 4.3 V 6 ND ND 1.7 2.7 ND ND <0.5 <0.5 V 7 2.1 4.0 15.6 27.3 1.4 3.0 10.2 14.6 V 8.1 + 8.2 1.2 1.9 8.7 17.3 0.6 1.2 5.1 6.8 V 8.2 1.5 2.6 12.0 23.1 0.7 1.3 6.0 7.2 V 9 ND ND <0.5 4.7 ND ND <0.5 <0.5 V 10 ND ND <0.5 4.1 ND ND <0.5 <0.5 V 14 ND ND 3.2 9.4 ND ND <0.5 <0.5 non-V 2, 7, & 8 ND ND 0.6 0.7 The fold increase in the absolute number of cells expressing indicated V s at the end of a 4-d culture period is shown for a range of concentrations of -GalCer and iGb3. Data are representative of four independent experiments. ND, not determined. Downloaded from jem.rupress.org on May 8, 2009 Published May 1, 2006 JEM VOL. 203, May 15, 2006 1203 ARTICLE thus faithfully mimicking the natural NKT cell bias. This highly selective eff ect was further illustrated by the near background CFSE dilution (57%) found for all the non-V 2, 7, 8 cells examined together at the highest concentration of iGb3 (Fig. 7, bottom right). Furthermore, iGb3 was 10 times more stimulatory for V 7 than for V 8.2 cells, as judged by the proportion of cells reaching the CFSE-negative stage and by the cell expansion measured in absolute numbers (Table I). For example, 40% of V 7 cells had reached the CFSE-negative stage at 0.5 ng/ml of iGb3 compared with only 34% of V 8.2 at 5 ng/ml (Fig. 7), and the level of expansion of V 7 at 0.5 ng/ml was not reached until 5 ng/ml for V 8.2 (Table I). Thus, the data establish a clear hierarchy of V stimulation by iGb3, with V 7>V 8.2>V 2, fully consistent with their respective 12-, 8-, and 2.6-fold enrichments during thymic selection (Fig. 2). This good correlation between the hierarchy measured in vitro and the selection bias in vivo is consistent with iGb3 being a major thymic ligand for NKT precursors and further suggests that positive selection rather than negative selection plays the primary role in shaping the V repertoire of V 14 NKT cells. Correlations between V selection and CD1d levels in vivo Previous reports have shown that changes in V selection occurred in mice expressing lower or higher levels of CD1d, Figure 7. V -specifi c hierarchy of responses to graded concentrations of antigens. CFSE-labeled V 14tg.CD1d / DN T cells were stimulated as in Fig. 6 using a range of antigen concentrations. Numbers in each histogram indicate the percentage of cells within the corresponding brackets of CFSE-negative (left) and CFSE-low (right) cells. Histograms with no percentages have <50% CFSE-diluted cells. Data are representative of four inde pendent experiments. Downloaded from jem.rupress.org on May 8, 2009 Published May 1, 2006 1204 V CONTRIBUTION TO GLYCOLIPID RECOGNITION BY NKT CELLS | Wei et al. and were hypothesized to result from changes in positive and negative selection according to V affi nities for naturally expressed ligands. Thus, in mice expressing very high levels of CD1d (50 times greater than WT) under a MHC class I promoter (13), NKT cells were decreased in absolute numbers to 1/3 of WT, suggesting negative selection, with V 2 NKT cells being relatively overrepresented compared with V 8 and V 7 cells. Conversely, V 7 were overrepresented in BALB/c.CD1d+/ mice as compared with WT (14). Although the intricacies of positive and negative selection and clonal competition preclude a defi nitive interpretation of such results when considered alone, the data are fully consistent with the hierarchy of V responsiveness to iGb3 and naturally expressed ligands as determined in the present study. Here, we present two additional CD1d expression models to further test this hierarchy in vivo. In pLck/high mice expressing CD1d under the control of the proximal Lck promoter in a NOD.CD1d / background, the level of CD1d was 12 times higher than that of WT mice (31). These Tg mice exhibited a dramatic decrease in the proportion of V 7+ NKT cells from 21 to 12%, without signifi cant changes in total cell numbers (Fig. 8). Although this Tg model was studied in the NOD background, which is known to be a low NKT expressor, the results were signifi cant because they could be compared with littermate controls in the very same genetic background. Conversely, in C57BL/6 mice expressing a CD1d null allele and a CD1d low allele (an unintended consequence of a loxP insertion in the 5 UTR of CD1d1 locus) (CD1d /LE mice), the percentage of V 7 cells reached 43% as compared with 22% in WT controls, with a corresponding decrease of V 8 cells from 57 to 41% (Fig. 8). Overall, NKT cell numbers were drastically reduced, suggesting that in the presence of a limited amount of ligands, positive selection could only rescue the highest affi nity cells. Thus, combining these two new models with the other two previously published, the in vivo thymic selection outcomes are remarkably consistent with the V hierarchy of affi nity directly established from our ligand stimulation assays. V -specifi c induction of CD69 upon intrathymic cell transfer To further study the recognition of naturally expressed NKT ligands, we transferred V nonbiased V 14-J 18 Tg DN T cells into the thymus of Ly5.1 congenic recipients. Cells were recovered from the host thymuses after 6 h and subjected to an analysis of V and CD69 expression. In the absence of exogenous antigen, CD69 was induced on a subset of transferred cells with a selective V 7 (46%)>V 8 (43%)>V 2 (38%) pattern (Fig. 9) reminiscent of iGb3 stimulation assays in vitro. Surprisingly, however, two additional V s, V 9 and V 14, exhibited signifi cant induction of CD69, albeit to lower levels than the canonical V 7, V 8, V 2, suggesting weaker signaling. This surprising result was consistently observed in 3/3 independent transfer experiments and may suggest that, in addition to iGb3, other thymic ligands may be able to activate NKT precursors. These putative antigens, however, must be unable to meet the critical signaling requirements for inducing cell expansion and/or survival. Unfortunately, the transferred V 14-J 18 Tg DN T cells could not be effi ciently tracked over the longer period of time required to evaluate cell proliferation, precluding a more complete assessment of the fate of these V 9 and V 14 cells. D I S C U S S I O N By transgenically expressing the V 14-J 18 transgene in a CD1d-defi cient background, we have created a DN T cell population with a TCR repertoire made of the canonical invariant chain of NKT cells combined with a broad, nonbiased V repertoire. Several features suggest that these Tg cells display a faithful representation of the preselection repertoire of TCR chains associated with V 14-J 18 chain. First, they originate in the thymus in the absence of selection by CD1d/ligand. Second, they express an endogenous TCR Figure 8. V repertoire changes in mice expressing high or low levels of CD1d. (top) Levels of CD1d expression (mean fl uorescence intensity indicated above histograms) by thymocytes of indicated mice. CD1d /LE are mice with a null ( ) allele and a low expression (LE) allele the result of a loxP insertion in the 5 UTR of CD1d1. pLck/high are mice with high level expression of a CD1d transgene driven by the proximal Lck promoter. (bottom) Percentages of V 7 and V 8 cells among -GalCer- CD1d tetramer+ cells were determined by FACS analysis of indicated mice. Bars represent mean and SD (white, V 7; gray, V 8) based on indicated n number of mice examined. CD1d = : relative CD1d expression level (mean fl uorescence intensity) compared with B6 WT mice; NKT = : relative numbers of NKT thymocytes compared with B6 WT mice. *, statistical signifi cance (P < 0.05) using unpaired Student s t test. Downloaded from jem.rupress.org on May 8, 2009 Published May 1, 2006 JEM VOL. 203, May 15, 2006 1205 ARTICLE repertoire of breadth and diversity comparable to that of nonselected DP thymocytes. Third, their ontogeny is related to the premature expression of the Tg TCR chain that binds to the TCR chains expressed at the DN3 stage. It is believed that, by modifying the signals that normally emanate from the pT TCR complexes, the TCR transgene bypasses the subsequent need for TCR-mediated positive selection (16 20, 32). Finally, similar to a subset of mature NKT cells, these Tg cells do not express CD4 or CD8. These Tg DN cells provide, therefore, a unique tool to explore the structure and the functional properties of the preselection repertoire of NKT precursor thymocytes and to contrast them with those of natural mature NKT cells. Our fi ndings elucidate some long-standing issues regarding the mechanisms underlying NKT cell repertoire selection. Because the nonselected V repertoire associated with V 14-J 18 diff ered only slightly from the one found in the nonselected DP thymocytes of MHC-null mice, which represents an average of all V s, we conclude that the V -pairing bias of V 14-J 18 chain must be modest compared with other V s. This fi nding contrasts with isolated or indirect evidence suggesting an inherent instability of many V 14 J 18/V complexes (10, 11). Second, by comparing the frequency of V families in the unselected and postselection repertoire, it was possible for the fi rst time to measure the degree of enrichment for individual families, with V 7 (x12) clearly above V 8.2 (x8) and V 2 (x2.6) at the bottom. The picture would be signifi cantly diff erent if the baseline for comparison was the DP repertoire because V 7 and V 8.2 would appear to be equally selected (14). Remarkably, these data matched precisely the hierarchy of V responses observed after stimulating the nonselected V 14-J 18 Tg T cells with naturally expressed ligands or with synthetic iGb3. Thus, we conclude that the V repertoire associated with V 14-J 18 in NKT lineage cells has undergone predominant positive selection and only modest negative selection. Importantly, the close resemblance between stimulation by iGb3 and stimulation by the naturally expressed NKT ligands provides new and independent support for the notion that iGb3 is the main NKT ligand involved in these thymic processes. A large proportion ranging from 20 to 50% of the V 8-, V 7-, and V 2-expressing cells responded to iGb3 in the CD69 induction assay, whereas nearly all of them responded in the CFSE dilution assay. Although the diff erent nature and kinetics of these assays may underlie these diff erences, the results converge to indicate that a substantial proportion of the naturally arising V 8, V 7, and V 2 TCRs may be selected during NKT cell development. Surprisingly, V 9- and V 14-expressing cells consistently exhibited some level of CD69 induction upon intrathymic transfer, despite the absence of response to iGb3 and despite the absence of corresponding cells in the natural NKT cell pool. This observation could indicate that additional unidentifi ed ligands partially activate NKT precursors during thymic development, yet may not promote their selection or expansion, perhaps as a result of lower affi nity. Another important insight gained from this study relates to the much larger repertoire elicited by synthetic -GalCer, a mimic of microbial glycuronosylceramides, than by endogenous ligands. Thus, many V s that are not stimulated by iGb3 and are not found in the natural NKT repertoire, including V 3, 6, 9, 10, 14, could nevertheless be stimulated by nanomolar concentrations of -GalCer. These unexpected fi ndings demonstrate that the NKT cell repertoire against foreign and microbial antigens is severely limited because thymic exposure to natural ligands failed to select potentially useful TCRs. This stands in sharp contrast with positive selection driven by polymorphic MHC ligands, where the opposite outcome is observed, namely a gain in recognition of foreign peptides after positive selection by self-ligands (33). We propose that these diff erences may be inherent to the processes of positive selection by partial agonists/ antagonists versus full agonists. Finally, our results directly confi rm and extend to the entire nonselected repertoire recent reports (14) suggesting that diff erent V families generically encode diff erent affi nities for lipid ligands. V 7 responded much better than V 8.2 to low doses of natural ligand, whereas both responded comparably to -GalCer and microbial ligands. Because of the absence of CDR3 restriction in these responses, the CDR1 and 2 regions of the chain may confer the differences in affi nity. Future structural studies of these interactions, including crystallography and mutational analysis, will Figure 9. V -specifi c induction of CD69 after intrathymic transfer of V 14tg.CD1d / DN T cells. DN T cells were intrathymically injected into Ly5.1 congenic host. After 6 h, CD69 expression was determined by FACS analysis of Ly5.2+CD4 CD8 -gated cells according to V expression. Data are means SD from three independent experiments. Downloaded from jem.rupress.org on May 8, 2009 Published May 1, 2006 1206 V CONTRIBUTION TO GLYCOLIPID RECOGNITION BY NKT CELLS | Wei et al. shed light on this intriguing mode of TCR recognition. In the meantime, our quantitative determination of the hierarchy of natural ligand recognition by diff erent V families provides potentially useful knowledge to assess the contribution of affi nity to the functional properties of NKT cell subsets and to the selection of NKT cells in mice exhibiting NKT cell defects. MATERIALS AND METHODS Mice. C57BL/6 and NOD mice were purchased from The Jackson Laboratory. C57BL/6. 2M / .I-Ab / (MHC-null) and C57BL/6.Ly5.1 congenic mice were purchased from Taconic Farm. C57BL/6.V 14tg and C57BL/6. CD1d / mice were described previously (15, 34), and were intercrossed to obtain C57BL/6.V 14tg.CD1d / mice. The CD1dLE (LE, low expression) allele was the unintended consequence of the insertion of a loxP site in the 5 UTR of the C57BL/6 CD1d1 gene after homologous recombination in ES cell and excision of a neomycinr gene and a STOP sequence cassette. This insertion resulted in the modifi cation of the genomic sequence upstream of the ATG site, 5 -T A G A A C T C T G G C G ^CTATG-3 , which included a deletion of the underlined GCG and an insertion at the position marked ^ of the sequence 5 -C A T A A C T T C G T A T A G C A T A C A T T A T A C G A AG T T A T A T T A A G G G T T A T T G A A T A T G A T C G G A A T T C C T C G A G CG G C -3 . The C57BL/6.CD1d /LE mouse carrying a null allele and a LE allele expressed very reduced levels of CD1d on the surface of thymocytes. The LE allele was screened using PCR (forward primer: 5 -T G T G T A GA A C T C T G C A T A A C T T C G -3 , reverse primer: 5 -G G G C C A G G T G T AT T C A G G A G C A T C T G C A C G G -3 ). All mice were housed in a specifi c pathogen-free facility at the University of Chicago according to the guidelines of the institutional animal care and use committee. Flow cytometry. Fluorochrome-conjugated mAbs anti-CD1d, CD3 , CD4, CD8 , CD44, CD69, Ly5.2, NK1.1, TCR , TCR V 2 through V 14, TCR V 2, V 3.2, V 8.3, and V 11.1+11.2, as well as biotinylated V 2 through V 14 mAbs, were purchased from BD Biosciences. Streptavidin conjugated to allophycocyanin (APC) or R-PE was purchased from eBioscience. Anti-V 8.2 mAb (clone F23.2) was biotinylated in our laboratory. CD1d tetramers loaded with -GalCer were prepared and used as described previously (23). CFSE labeling of enriched splenocytes was done in vitro according to manufacturer s instructions in CellTrace CFSE Cell Proliferation kit (Invitrogen). Flow cytometry analysis was performed on a four-color FACSCalibur with CELLQuest software (BD Biosciences). CDR3 sequencing. DN T cells were sorted as TCR +CD4 CD8 cells from C57BL6.V 14tg.CD1d / splenocytes. RNA extraction, reverse transcription PCR, and plasmid cloning and sequencing methods were as described previously (35). Primers were 5 -C C T C A T T C T G G A G T T G G C T A C C C -3 for V 8.2+8.1, 5 -G A A G A T G G T G G G G C T T T C A A G G A T C -3 for V 12, and 5 -C T T G G G T G G A G T C A C A T T T C T C -3 for C . CDR3 sequence analyses were performed according to the international ImMunoGeneTics (IMGT) information system website (http://imgt.cines.fr) (36). Cell stimulation assays. Single cell suspensions prepared from four to fi ve adult C57BL6.V 14tg.CD1d / spleens were fi rst enriched for DN T cells by depleting B, CD4, CD8, and NK cells with B220, CD4, CD8 , and DX5 mAb-conjugated magnetic microbeads and AutoMACS (Miltenyi Biotec) according to manufacturer s instructions. DX5 is not expressed by the DN T cells. For the CFSE expansion assay, this enriched population (>50% were DN CD3 + cells) was labeled with CFSE before stimulation with lipid-pulsed BM-DCs. The DCs were prepared from cultures of BM cells in a 1:1 mixture of EHAA and RPMI 1640 (Biofl uids) supplemented with 10% heat-inactivated fetal calf serum, glutamine, antibiotics, 5 10 5 M of 2-mercaptoethanol and 2 ng/ml of mouse GM-CSF (Biosource). After 6 d of culture, BM-DCs were pulsed with diff erent concentrations of -GalCer or iGb3 overnight, washed, and plated at 106 per well in six-well plates (Corning Costar). 10 106 enriched DN T cells were added per well and cultured in the presence of mouse recombinant IL-7 at 10 ng/ml (Biosource) for 4 d before FACS analysis. For the CD69 induction assay, the lipid-pulsed DCs were allowed to adhere to culture wells for 16 24 h before adding the enriched DN T cells. These cocultures were spun down briefl y (1,200 revolutions/min) to favor cell-to-cell contact. After 4 6 h, the DN T cells were harvested and processed for FACS analysis, using anti-CD69, V s, CD3 , and CD4 and CD8 antibodies. Intrathymic cell transfers. DN T cells enriched from C57BL/6.V 14tg. CD1d / spleens (Ly5.2) by depleting B, CD4, CD8, and NK cells with B220, CD4, CD8 , and DX5 mAb-conjugated magnetic microbeads and AutoMACS were injected intrathymically into C57BL6.Ly5.1 congenic hosts as described previously (37). After 6 h, thymocytes were harvested and depleted of CD4- and CD8 -expressing cells using magnetic microbeads and AutoMACS (Miltenyi Biotec) before FACS analysis. Transferred DN T cells cells were identifi ed using anti-Ly5.2, CD69, V s, and CD4 and CD8 antibodies. We thank B. Jabri for expert advice in TCR repertoire analysis,Copyright Borowski and K. Griewank for helpful discussions, and all members of the Bendelac Lab for help and support. This work was supported by National Institutes of Health grants to A. Bendelac (no. AI038339) and A. Bendelac, P.B. Savage, and L. Teyton (no. PO1 AI053725). The authors have no confl icting fi nancial interests. Submitted: 21 February 2006 Accepted: 27 March 2006 R E F E R E N C E S 1. Godfrey, D.I., H.R. 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Kaufmann, and U.E. Schaible. 2004. Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells. Proc. Natl. Acad. Sci. USA. 101:10685 10690. 10. Gui, M., J. Li, L.J. Wen, R.R. Hardy, and K. Hayakawa. 2001. TCR chain infl uences but does not solely control autoreactivity of V 14J281T cells. J. Immunol. 167:6239 6246. Downloaded from jem.rupress.org on May 8, 2009 Published May 1, 2006 JEM VOL. 203, May 15, 2006 1207 ARTICLE 11. Degermann, S., G. Sollami, and K. Karjalainen. 1999. Impaired NK1.1 T cell development in mice transgenic for a T cell receptor chain lacking the large, solvent-exposed c FG loop. J. Exp. Med. 190:1357 1362. 12. Schumann, J., R.B. Voyle, B.Y. Wei, and H.R. MacDonald. 2003. Cutting edge: infl uence of the TCR V beta domain on the avidity of CD1d: -galactosylceramide binding by invariant V 14 NKT cells. J. Immunol. 170:5815 5819. 13. Chun, T., M.J. Page, L. Gapin, J.L. Matsuda, H. Xu, H. Nguyen, H.S. Kang, A.K. Stanic, S. Joyce, W.A. Koltun, et al. 2003. CD1d-expressing dendritic cells but not thymic epithelial cells can mediate negative selection of NKT cells. J. Exp. Med. 197:907 918. 14. Schumann, J., M.P. Mycko, P. Dellabona, G. Casorati, and H.R. Macdonald. 2006. Cutting edge: infl uence of the TCR V domain on the selection of semi-invariant NKT cells by endogenous ligands. J. Immunol. 176:2064 2068. 15. Bendelac, A., R.D. Hunziker, and O. Lantz. 1996. Increased interleukin 4 and immunoglobulin E production in transgenic mice overexpressing NK1 T cells. J. Exp. Med. 184:1285 1293. 16. von Boehmer, H., J. Kirberg, and B. Rocha. 1991. An unusual lineage of / T cells that contains autoreactive cells. J. Exp. Med. 174:1001 1008. 17. Bruno, L., H.J. Fehling, and H. von Boehmer. 1996. The T cell receptor can replace the receptor in the development of lineage cells. Immunity. 5:343 352. 18. Terrence, K., C.P. Pavlovich, E.O. Matechak, and B.J. Fowlkes. 2000. Premature expression of T cell receptor (TCR) suppresses TCR gene rearrangement but permits development of lineage T cells. J. Exp. Med. 192:537 548. 19. Borowski,Copyright, X. Li, I. Aifantis, F. Gounari, and H. von Boehmer. 2004. Pre-TCR and TCR are not interchangeable partners of TCR during T lymphocyte development. J. Exp. Med. 199:607 615. 20. Baldwin, T.A., M.M. Sandau, S.C. Jameson, and K.A. Hogquist. 2005. The timing of TCR expression critically infl uences T cell development and selection. J. Exp. Med. 202:111 121. 21. Caveno, J., Y. Zhang, B. Motyka, S.J. Teh, and H.S. Teh. 1999. Functional similarity and diff erences between selection-independent CD4 CD8 T cells and positively selected CD8 T cells expressing the same TCR and the induction of anergy in CD4 CD8 T cells in antigen-expressing mice. J. Immunol. 163:1222 1229. 22. Zerrahn, J., W. Held, and D.H. Raulet. 1997. The MHC reactivity of the T cell repertoire prior to positive and negative selection. Cell. 88:627 636. 23. Benlagha, K., A. Weiss, A. Beavis, L. Teyton, and A. Bendelac. 2000. In vivo identifi cation of glycolipid antigen-specifi c T cells using fl uorescent CD1d tetramers. J. Exp. Med. 191:1895 1903. 24. Kato, T., H. Sasakawa, M. Kurokawa, K. Masuko-Hongo,Copyright Hamada, K. Yamamoto, and K. Nishioka. 1999. Comparison of T-cell receptor J gene usage in spleen cells of diff erent mouse strains. Microbiol. Immunol. 43:93 97. 25. Wallace, M.E., M. Bryden, S.C. Cose, R.M. Coles, T.N. Schumacher, A. Brooks, and F.R. Carbone. 2000. Junctional biases in the naive TCR repertoire control the CTL response to an immunodominant determinant of HSV-1. Immunity. 12:547 556. 26. Hsieh, C.S., Y. Liang, A.J. Tyznik, S.G. Self, D. Liggitt, and A.Y. Rudensky. 2004. Recognition of the peripheral self by naturally arising CD25+ CD4+ T cell receptors. Immunity. 21:267 277. 27. Pannetier,Copyright, M. Cochet, S. Darche, A. Casrouge, M. Zoller, and P. Kourilsky. 1993. The sizes of the CDR3 hypervariable regions of the murine T-cell receptor chains vary as a function of the recombined germ-line segments. Proc. Natl. Acad. Sci. USA. 90:4319 4323. 28. Matsuda, J.L., O.V. Naidenko, L. Gapin, T. Nakayama, M. Taniguchi, C.R. Wang, Y. Koezuka, and M. Kronenberg. 2000. Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers. J. Exp. Med. 192:741 754. 29. Moody, D.B., V. Briken, T.Y. Cheng,Copyright Roura-Mir, M.R. Guy, D.H. Geho, M.L. Tykocinski, G.S. Besra, and S.A. Porcelli. 2002. Lipid length controls antigen entry into endosomal and nonendosomal pathways for CD1b presentation. Nat. Immunol. 3:435 442. 30. Rabinowitz, J.D.,Copyright Beeson,Copyright Wulfi ng, K. Tate, P.M. Allen, M.M. Davis, and H.M. McConnell. 1996. Altered T cell receptor ligands trigger a subset of early T cell signals. Immunity. 5:125 135. 31. Wei, D.G., H. Lee, S.H. Park, L. Beaudoin, L. Teyton, A. Lehuen, and A. Bendelac. 2005. Expansion and long-range diff erentiation of the NKT cell lineage in mice expressing CD1d exclusively on cortical thymocytes. J. Exp. Med. 202:239 248. 32. Eberl, G., H.J. Fehling, H. von Boehmer, and H.R. MacDonald. 1999. Absolute requirement for the pre-T cell receptor chain during NK1.1+ TCR cell development. Eur. J. Immunol. 29:1966 1971. 33. Bevan, M.J. 1977. In a radiation chimaera, host H-2 antigens determine immune responsiveness of donor cytotoxic cells. Nature. 269:417 418. 34. Chen, Y.H., N.M. Chiu, M. Mandal, N. Wang, and C.R. Wang. 1997. Impaired NK1+ T cell development and early IL-4 production in CD1-defi cient mice. Immunity. 6:459 467. 35. Park, S.H., A. Weiss, K. Benlagha, T. Kyin, L. Teyton, and A. Bendelac. 2001. The mouse CD1d-restricted repertoire is dominated by a few autoreactive T cell receptor families. J. Exp. Med. 193:893 904. 36. Lefranc, M.P., V. Giudicelli, Q. Kaas, E. Duprat, J. Jabado-Michaloud, D. Scaviner,Copyright Ginestoux, O. Clement, D. Chaume, and G. Lefranc. 2005. IMGT, the international ImMunoGeneTics information system. Nucleic Acids Res. 33:D593 D597. 37. Benlagha, K., D.G. Wei, J. Veiga, L. Teyton, and A. Bendelac. 2005. Characterization of the early stages of thymic NKT cell development. J. Exp. Med. 202:485 492. Downloaded from jem.rupress.org on May 8, 2009 Published May 1, 2006
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Dana Wilkey Shows Us Her Digs In Beverly Hills
Wonder what it's like to live the life of luxury like The Real Housewives of Beverly Hills? Join SheKnows as the cast's newest member Dana Wilkey takes us on a private home tour!
SheKnows got the privilege of touring the home of the new Real Housewives of Beverly Hills cast member before anyone else. Wilkey only recently bought the lavish 16,000-square-foot hilltop house, and it's yet to be featured on the show. It's all about entertainingBoasting eight bedrooms, 13 bathrooms and multiple stocked bars, this home was built for fun. Wilkey showed us her game room, equipped with a pool table, a shuffleboard, a Baywatch pinball machine and an old-school Pac-man game. Outside, the party-ready pad includes a comfy rooftop cabana for stargazing and a ground-level poolside cabana for summer fun. But the icing on this celebratory cake is what the pool can do: Wilkey told us the yellow glint it gives off is from the discotheque lighting system that can be turned on for parties. Kicking backBut this businesswoman isn't always in the mood to be "on." When she wants to unwind, Wilkey heads for her steam room and sauna. The relaxing rooms are located conveniently across from her enormous wine cellar, the perfect combo after a long day of watching housewives catfight. And what would a reality star's home be without a screening room? Dana decked out her home theater with her favorite movie posters and a nostalgic popcorn machine. Conner, her soon-to-be stepson, uses the room to practice his music. Photo gallery: More photos of Dana's home >>More celebrity home décorCelebrity décor: Sharon and Ozzy Osbourne's Los Angeles home


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It was the best experience I ever had at a hospital.
There are times when Nancy Phenix can't find where she put her cane. She doesn't need it much anymore.
It's a remarkable turn-around considering that just four months earlier she had spent a month in Borgess Medical Center after having a major stroke at her home in Coldwater. The 63-year-old retired Michigan State Police Officer credits a large part of her recovery to the skills and attitude of the staff at the Borgess Neuro Rehabilitation program.
"The therapy was intense but wonderful," said Phenix. "It was the best experience, all things considered, I ever had in a hospital."
Phenix, who lives alone, fell to the floor the day of the stroke, which paralyzed her left side, and couldn't get back up. "I scooted my body a little at a time from one end of the house to the other to get to the phone. I knew I could make it. I don't scare easily."
She called a neighbor who called for an ambulance. Taken to Coldwater Hospital she was transferred to Borgess where she was later moved to the Neuro Rehab unit.
"The staff at the Neuro Rehab program challenged me to the point where I'd say, 'You've got to be kidding.' They weren't kidding. Everyone on the floor was absolutely great. The care was perfect."
Phenix said that the rehabilitation included physical, occupational and recreational therapy. They had her do laundry and cook. "They knew I live alone and didn't want me to go home until I could do things for myself.
"I am so thankful to them."
Phenix said that she was able to drive short distances within a few months of returning home. She now offers her time for peer support to other stroke survivors.
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The football scenes were shot at Los Angeles' Gilmore Stadium, the current site of CBS Television City, with the Loyola University football team. Gilmore location details with then-and-now photos are reported in The Three Stooges Journal # 108 (Winter 2003). The opening street sequences with the Stooges were filmed on Yucca and Vine Streets in downtown Hollywood. The details with then-and-now photos are published in The Three Stooges Journal # 103 (Fall 2002). Larry said that there were numerous injuries while this short was made, including him losing a tooth when he was socked by Joseph Young (actor Robert Young's brother), and Curly broke his leg after riding down the dumbwaiter. In the scene with the photographers during the football game, Larry insisted the studio use stunt doubles, one of whom was badly injured. The original ending had the Stooges retelling the story to their children, but only photo stills remain of that footage (see Moe Howard & the 3 Stooges pp. 6-7, 68-69, 79 and 81). The complete, original scripted version with the Stooges' sons, was published in The Three Stooges Book of Scripts, by Joan Howard Maurer, from Citadel Press in 1987. Opening title music is "Eastmoor College March," by Louis Silvers; see The Three Stooges Journal # 87 (Fall 1998). |
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Title Origin: | Three Little Pigs (1933 Disney cartoon) |
Prod. No.: | 156 |
Shooting Days: | 5 days From: 1934-10-25 To: 1934-10-30 |
Face Slaps: 30 | Eye Pokes: 1 | Head Bonks: 0 | Pastry Thrown: 0 |
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One of the best of the earliest Stooge shorts, in my opinion. Three Little Pigskins offers up a lot of bangs for your buck.
Final Shemp's Final Word: 4 Pokes
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IDG News Service - Facebook has fixed a flaw that let hackers delete Facebook friends without permission.
The flaw was reported Wednesday by Steven Abbagnaro, a student at Marist College in Poughkeepsie, New York. It was patched Friday afternoon, Pacific time, after the IDG News Service notified Facebook of the issue.
The bug was a variation of an earlier vulnerability that Facebook learned about last week, which affected a range of features on the Web site. Hackers could have leveraged Abbagnaro's bug to delete all of a victim's contacts, one by one, but it does not appear that anyone ever exploited it in a malicious way.
For Abbagnaro's attack to work, however, a user would have to have been tricked into clicking on a malicious Web link while still logged into Facebook.
Facebook has struggled this week to fix these bugs, which are called cross-site request forgery flaws. They exist because of relatively simple Web programming mistakes in the Web site's code, and security researchers have criticized Facebook for not fixing them more quickly.
"We're in the process of doing a full audit and are building additional protections for this type of potential attack across the code base," said Simon Axten, a Facebook spokesman, in a Friday e-mail interview. "We began working on this one as soon as we learned about it and pushed a fix early this afternoon."
Robert McMillan can be reached at firstname.lastname@example.com. He is on Twitter at: http://twitter.com/bobmcmillan
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text: One of Zeus's daughters has been whispering in my ear, so quietly and soft she must be very near. Her unexpected presence to leave a word or clever phrase, is genuinely welcome because for them, I get the praise! People tell me that I'm good and I smile contentedly, waiting for the muse again and the words she'll leave for me.


text:
Feel free at ringtone download service Audiko.net, where it's possible to search for free ringtones for your Android smartphone. Today we have a ringtone of "Job" by Curren$y as well as 1251 other ringtones by Curren$y. "Job" is a real hit from the "Verde Terrace" album that was composed by Curren$y at their studios together with other 23 tracks. You can listen to all 7 Curren$y – "Job" ringtone variations available here and download it for free to your PC. You can also find other 10 albums produced by Curren$y for all time. "Job" is referred to Rap, and we bring you a huge amount of Rap melodies absolutely for free. And finally, we have collected lyrics for this track and you can view it here as well. So feel free to browse, listen, download ringtones at Audiko.net. Make your phone sound cool!
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2br, Williamsburg, Brooklyn, $5,000
Spacious apartments with upscale details, incredible amenities, and a location that surrounds you with stunning views and exciting venues.The excitement of living on the East River waterfront begins with this spectacular building's exterior, distinguished by the undulating floor-to-ceiling bay windows that provide most apartments with panoramic views of East River State Park, East River and Manhattan skyline.
Condo-quality finishes abound here. The living spaces feature expansive layouts, high ceilings and unending views. The restaurant-sized kitchens feature lacquer and wood custom cabinetry and a full-suite of Bosch and Kitchen Aid stainless steel appliances. The over-sized bathrooms rival elite spas with custom dual-sink vanities, deep soaking tubs and radiant-heat flooring.
The amenities are premier. Furnished and attended lobby. The eighth-floor roof deck is a scene-maker with a pool and lounge. The landscaped roof deck offers endless city views and a large space for al fresco entertaining. A courtyard provides respite from the city rush. The high-end fitness center features cardio and weight equipment as well as a yoga studio. A high-tech resident lounge offers a 60" flat-screen TV wired with DIRECTV(r). Indoor parking and storage spaces are also available for lease.
You cannot ask for a better location! The newly created East River State Park is literally your front yard. On surrounding streets, Williamsburg's most fashion-forward shops and boutiques offer endless opportunities for indulging. A three-block walk takes you to Bedford Avenue for nightlife as well as the L train to Manhattan. There's also Manhattan ferry service available across the street.
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Dishwasher, Doorman, Elevator, Gym, Laundry Room, On-site super, Outdoor Space, Parking, Pool, Storage Room
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text:
"Exactly what I was looking for!"
4.5 starson by djdybwadPros: Large screen, good touch-screen controls, seems to do it all
Cons: Battery life is marginal for a two-hour movie, Opera needs some work on ease-of-use
Summary: I really love the Apple iPod Touch, but the screen is too small for my eyesite. The Archos 705 and it's predecessor the 704 offer a large screen for those who really want to watch a movie without eye strain.
The 705 acts just like an external hard drive as far as Windows Explorer is concerned. You can drag-and-drop from your PC to the Archos.
You can also use Windows Media Player to keep your PC and the 705 in perfect sync.
In addition to photos, music and video, I also backup my company files onto the Archos. No, I can't run my applications here, but my backups are safe.
I purchased the 705 to allow me to surf the web while watching my TV. Standard web pages are very readable but response is slow. I wish it used an "N" wireless connection instead of a "G" connection.
The pop-up typewriter allows the user to use a finger to point out the letters in a URL search or in an E-mail. The response is too cumbersome to write a novel, or even a one-page E-mail.
Battery life has been about 20% than I expected from the Archos specifications. It has a five-element battery life indicator, but the last two elements display for only minutes on the screen and then it's time to power off.
I purchased the Opera browser which works OK. A "back" button (previous screen) needs to be put into the top browser bar, instead of having to click on menu>navigation>back.
I do carry the Archos 705 back and forth to work every day in the supplied case, and I'm 95% pleased with it.


text: Guidelines for Consumer-focused Public Reporting (Text Version) Slide presentation from the AHRQ 2008 conference showcasing Agency research and projects. Slide Presentation from the AHRQ 2008 Annual ConferenceOn September 10, 2008, Carol Cronin, made this presentation at the 2008 Annual Conference. Select to access the PowerPoint® presentation (82 KB; Plugin Software Help).Slide 1Guidelines for Consumer-focused Public ReportingAHRQ Quality Indicators (QI) Users MeetingCarol CroninInformed Patient InstituteSeptember 10, 2008Slide 2BackgroundNational Quality Forum (NQF) Public Reporting Technical Advisory Panel (TAP)?Part of NQF Steering Committee (SC)—Hospital Care 2007 project.AHRQ-funded.Slide 3TAP Charge"Recommend a Web-based approach for public reporting of acute hospital quality data that can be used, at minimum, to report the AHRQ Quality Indicators."Slide 4ScopeFocuses on reporting healthcare quality data from acute care hospitals in a Web-based format; though generalizable to other types of settings.Intended primarily for use by sponsors of consumer-focused sites to enable reports that support consumer understanding and participation in care decisions.Slide 5TAP MembersChair: Carol Cronin-Informed Patient Institute, Annapolis, MD.Katherine Brown, Hospital Quality Alliance, Washington, DC.Susan Dragoo, INTEGRIS Health, Oklahoma City, OK.Judy Hibbard, University of Oregon, Eugene, OR.Art Levin, Center for Medical Consumers, New York, NY.Denise Love, National Assoc. of Health Data Orgs., Salt Lake City, UT.Jeanne McGee, Mcgee & Evers Consulting, Vancouver, WA.Deirdre Mylod, Press Ganey Assoc., Inc., South Bend, IN.Ramesh Sachdeva, Children's Hospital and Health System, Milwaukee, WI.Maribeth Shannon, California HealthCare Foundation, Oakland, CA.Bruce Spurlock, CA Hospital Assesment & Reporting Taskforce, Roseville, CA.Project Staff: Melinda Murphy-NQF Consultant.Slide 6ApproachLiterature review.Review of existing Web-based resources—Talkingquality.gov, Usability.gov.Structured interviews with researchers, report sponsors.Iterative development.Slide 7AHRQ Model ReportDesigned to report comparative hospital QIs.Developed by researchers including Shoshanna Sofaer—Baruch College.Included testing with consumers.Two resulting model reports: Health Topics—individual QI indicators grouped into categories.Composites—QI composites.Slide 8Report Sponsor ChallengesUnderstanding what constitutes a useful report.Assuring reports support consumer understanding of quality and healthcare choice.Conveying information that provides the right amount of information at the right time in a way that resonates with target audiences.Customization of results to the audience.Slide 9TAP PrinciplesPublic and other stakeholders have right to access objective measures of quality provided by organizations in/from which they receive care, deliver care, purchase care, or provide funding/regulation and to receive the information in an understandable format.Use of the public reporting guidance can/should provide incentives for quality improvement.Information should be displayed for an array of common, cross-cutting conditions, all ages and be available across organizational settings/service lines and over time.Awareness and understanding of inherent values and biases is important to responsible reporting.Slide 10Goals of Consumer-focused Public ReportingIncrease consumer motivation to use public reports by making reports more understandable and relevant.Provide objective, unbiased, actionable and evaluable performance information to the public.Improve quality of care provided across the industry; andStimulate further evolution of the quality and comparability of public reporting at the organization, state and national levels.Slide 11Guidance for Consumer-focused Public Reporting—OverviewTAP identified guidelines for public reporting in 7 areas.Also included Implementation Considerations that amplify the guidelines.Assessed AHRQ Model Reports against guidelines.Includes AHRQ Model reports in Appendix.Slide 12Guideline 1: Purpose of Web-based ReportIdentify the purpose of the Web-based report, its intended main consumer audience(s), and how the report will be made known to the audience. Nature and purpose.Who is the audience? What are their info needs?Who are secondary audiences and how will their unique needs be addressed?Slide 13Guideline 2: Process of Developing the Web-based ReportDevelop the Web-based report using a transparent process that involves consumers and other relevant stakeholders. Stakeholders include: sponsors, consumers, organizations being measured.Establish governance/decision making rules.Opportunity for those being measured to preview and correct errors.Consumer usability testing before, during, after.Slide 14Guideline 3:Introduce concept of qualityAt the beginning of the report, set the stage by communicating what quality is, how quality varies and how making quality comparisons can be of value to consumers. Explain that quality varies within and across orgs.Use consistent, simple and familiar language.Slide 15Guideline 4: Meaningful MeasuresEnsure that measures included are meaningful to consumers, transparent, and meet widely accepted, rigorous criteria including importance, scientifically acceptable, feasible and usable. Relevant to consumers.Demonstrate variation.Provide information that reflects overall quality.Slide 16Guideline 5: Data PresentationPresent and explain data clearly and objectively in ways that help consumers understand and use it. Data should be evaluable.Consistent reporting (low or high scores better)�Use stories to illustrate meaning.Allow users to see what they want.Slide 17Guideline 5: Data Presentation (cont.)In presenting comparative quality information. Use tools such as rank ordering, symbols that help user quickly discern performance.Include benchmarks if possible.Provide risk-adjusted rates into categories using words such as "better", "average."Label indicators in everyday language.Limit use of statistics that are difficult for consumers to understand.Slide 18Guideline 5: Data Presentation (cont.)In presenting information from composite measures: Report all measures that comprise the composite.In providing contextual/decision support: Provide clear contextual information.Provide clear explanation for missing data.Consumer test.Use reasonably current data and display dates covered.In presenting technical documentation: Include detailed measure definitions, specs etc.Provide details about the methodology.Slide 19Guideline 6: Report UsabilityEnsure that design and navigation features enhance report usability. Organize information to let users make choices.Provide engaging format/easy navigation.Easy to skim.Easy to print.Test with audience.Slide 20Guideline 7: Regularly Review and Improve ReportsRegularly review and assess reports to ensure their effectiveness and currency. Assess use and impact.Involve stakeholders in revisions.Use learnings to drive improvement and usefulness of performance measures and public reporting field.Slide 20Research RecommendationsImpact of public reporting.Content of public reporting.Unintended consequences.Understanding issues related to cultural and linguistic needs.Effect of public reporting on quality improvement.Slide 22Project StatusPublic comment period: June 2008.Final NQF member voting: Aug. 2008.Consensus Standards Approval Committee (CSAC)/Board consideration: Sept. 2008.Publicly available: Late 2008.Slide 23For more information:National Quality Forum: www.qualityforum.org. "Guidelines for Consumer-focused Public Reporting."AHRQ Model Reports Send request to: firstname.lastname@example.com Quality: https://talkingquality.ahrq.gov Current as of February 2009 Internet Citation: Guidelines for Consumer-focused Public Reporting (Text Version). February 2009. Agency for Healthcare Research and Quality, Rockville, MD. http://www.ahrq.gov/news/events/conference/2008/Cronin.html


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Leading Innovative OrganizationsFaculty Coordinator: Terri Griffith Students taking this concentration must complete:
In addition, students must complete 12 additional credits from the following courses to deepen understanding of innovation, leadership in complex environments, team and project management, and decision-making skills for success in rapidly-changing environments:
Note: Students wishing to emphasize Innovation should include at least some of the courses marked **. |
Please click here for the most up to date information on this concentration. |


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I remember reading that in addition to Cuban Havanna Club the cuba libre drink tastest the best with mexican coca cola.
We've been to Cuba a few times and on the first trip I bought a 15-year-old Havanna Club from Havanna -- I was wondering a bit about the steep price. That rum was actually made in the US (and sold in Cuba), I later bought a real cuban 15-yo Havanna Club from Spain.


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04 Dec 2011 | #1 |
| what is the better options to use to do incremental backup Hi, I was trying to back up data from office to home via VPN connection. All PCs running Windows 7 for both ends. I have a batch file that uses Robocopy to perform "incremental backup" from the office to home. Here is the example robocopy c:/sharedFiles "\\192.168.1.10\myHome\sharedFiles" /E When I run the batch file, the VPN connection was fine. So the VPN was not the issue. I found it strange that every time I run the batch file. It uploaded the same file again and again. I was pretty sure it already did it but it marked it as newer and did upload the file again and again. This is a waste of bandwidth. Wonder what's the best way to do incremental backup? I am tempted to use /mir but this option is very risky as I could have important files deleted by accident. By my understanding, option /E should do the job as it only copies the modified files as well as new files and empty folders. That's what I want to do. Any help would be greatly appreciated. Thank you |
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04 Dec 2011 | #2 |
| I would purchase an external hard drive and do the backup locally, in the office using Acronis True Image or Macrium Reflect Free. Acronis also supports network backups but that has got to be slow compared to backing up to a local USB external drive. As for Robocopy, I think you need to use the /XO flag along with /E. /XO : eXclude Older - if destination file exists and is the same date or newer than the source - don't bother to overwrite it. |
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04 Dec 2011 | #3 |
| To sync data files, I would use Allway Sync. It is easy, fast and reliable. |
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04 Dec 2011 | #4 |
| Hi, See if this tutorial helps you: The /MIR option is probably the want you want, but as you recognise it can be dangerous. ROBOCOPY - Create Backup Script Regards, Golden |
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04 Dec 2011 | #5 |
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Top leaders in the Democratic Party called on embattled Rep. Anthony Weiner (D-N.Y.) to resign Saturday even as the scandal-tarred congressman announced he would request a leave of absence from the House to seek an unspecified form of treatment.
Weiner spokeswoman Risa Heller said Saturday afternoon that the congressman "departed this morning to seek professional treatment to focus on becoming a better husband and healthier person." She added that he would request "a short leave of absence from the House," after which he would make a decision on his political future. Heller did not return an e-mail regarding what sort of treatment Weiner would undergo.


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DARET EZZA, Syria: The rebel-held communications centre at Daret Ezza in northwest Syria is an archaeologist's worst nightmare, consisting of machine-gunned and burnt-out concrete bunkers perched atop ancient Byzantine ruins.
President Bashar al-Assad's army held the strategic telecoms station until it was captured by rebels, who use the position to scour the skies for regime helicopters and warplanes coming to bombard targets in the region.
Three months ago Free Syrian Army fighters took the position at the summit of a rocky mountain range 28 kilometres (17 miles) from Aleppo which offers a clear view from the Turkish border all the way to the crucial northern metropolis.
Thanks to information from two defecting Syrian soldiers, the heavily defended outpost fell within a few hours, with rebels losing four "martyrs" but killing 60 troops, says Ahmad, an Al-Faruq Brigade fighter who took part in the assault.
"Dushka (heavy Russian-made) machineguns had been set up" among the ruins of 10 Byzantine towers, he says, pointing at the circular collections of large stone blocks covered in rubble and blackened by flames.
Next to nothing now remains of the ancient Byzantine citadel at the mountain summit. Imposing white stone monoliths barely blemished by centuries of wear now serve as the foundation for hideous concrete bunkers.
Small ancient fortresses, some of which contain the entrances to underground tunnels previously shut to visitors, have been burned out. A rectangular building has collapsed in on itself from the blast of an explosion.
A twisted metal frame is all that remains of another building. An unexploded bomb the size of a fridge that has fallen from the sky sits on a low wall. It has been defused.
Blacked-out carcasses of Russian-made military trucks and destroyed radar stations also bear witness to the ferocity of the fighting.
The enormous satellite dishes have disappeared, probably removed by rebels to set up makeshift communications systems.
The Daret Ezza post, nearly 12 kilometres from the Turkish border, was an army observation and listening station supported by Russian technicians, some rebels claim. There is of course no longer any trace of the Russians.
Below the post, a mobile phone mast has miraculously survived the destruction and appears to be still working.
The rebels shelter at night from the wind in the bunkers, which lead underground, coming out during the day to monitor the skies through binoculars, informing their commanders of every aircraft that passes overhead.
The Syrian army has lost its control over most of the region, now holding only two garrisons both of which are surrounded by rebels.
One is at Atareb, 12 kilometres west of Aleppo, and another is at Sheikh Suleiman, 25 kilometres to the northwest.
The capture of the two remaining regime bastions, which rebels say is imminent, will enable the almost total "liberation" of the northwestern region that runs the length of the border with Turkey.