Loss of Ly-6A.2 expression on immature developing T cells in the thymus is necessary for their normal growth and generation of the Vbeta T-cell repertoire.

Stage-specific expression of a number of cell-surface and signaling proteins is critical for normal development of T cells in the thymus. Equally important may be the loss of expression/signaling of developmentally regulated proteins for proper transitioning of developing T cells into thymic subsets. Ly-6A.2 exhibits a regulated pattern of expression on T cells maturing in the thymus, and dysregulating its expression results in arrest of developing T cells within the CD3-CD4-CD8- triple negative (TN) stage where the normal expression of Ly-6A.2 is extinguished. To further characterize the mechanisms underlying this block, we examined whether cell signaling and/or cell adhesion properties of the Ly-6A.2 molecule influenced the block in T-cell development. Analysis of bone marrow chimeras generated by injecting CFSE-labeled Ly-6A.2 transgenic bone marrow cells into irradiated syngeneic non-transgenic mice revealed normal trafficking of developing T cells from the cortex into the medulla. Production of LAT but not p56lck was diminished in CD4-CD8- DN cells from Ly-6A.2 dysregulated mice when compared with control littermates. Dysregulated expression of Ly-6A.2 did not suppress endogenous TCR-Vbeta expression. Finally, dysregulated expression of Ly-6A.2 enhanced apoptosis of an immature CD4+CD8+ (DP) subset of developing cells and altered the selected TCR-Vbeta repertoire. Taken together, these observations indicate that the termination of Ly-6A.2 expression and signaling within the CD4-CD8-CD3- subset of developing T cells is an important checkpoint during normal thymic development.

Downregulated expression of Ly-6-ThB on developing T cells marks CD4+CD8+ subset undergoing selection in the thymus.

Interaction of TCRs on CD4+CD8+ immature T cell with MHC-peptide complexes on stromal cells is required for positive and negative selection in the thymus. Identification and characterization of a subpopulation of CD4+CD8+ thymocytes undergoing selection in the thymus will aid in understanding the mechanisms underlying lineage commitment and thymic selection. Herein, we describe the expression of Ly-6 ThB on developing thymocytes. The majority of CD4+CD8+ thymocytes express Ly-6 ThB at high levels. Its expression is downregulated in a subset of CD4+CD8+ thymocytes as well as in mature CD4+CD8- and CD4-CD8+ T cells. More importantly, interaction of TCR/coreceptor with the self-MHC-peptide contributes to the downregulation of ThB expression on developing thymocytes. These findings indicate that downregulation of ThB on CD4+CD8+ thymocytes identifies a unique subset (CD4+CD8+ThBneg-low) of thymocytes that has received the initial signals for thymic selection but have not yet downregulated the CD4 and CD8 cell surface expression. In addition, these results also indicate that a high frequency (approximately 20-40%) of CD4+CD8+ immature thymocytes receive these initial signals during thymic selection.

A monoclonal antibody against the 66-kDa protein expressed in mouse spleen and thymus inhibits Ly-6A.2-dependent cell-cell adhesion.

The Ly-6 locus encodes several cell surface proteins of 10-12 kDa. Some members of this multigene family may function in cell signaling and/or cell adhesion processes. T lymphocytes overexpressing Ly-6A.2 (one member of the Ly-6 gene family) protein homotypically aggregate when cultured in vitro. Further analysis of this homotypic aggregation suggests that Ly-6A.2 participates in cell-cell adhesion. These observations indicated the presence of a Ly-6 ligand(s) on the surface of lymphoid cells. In this study we report generation of a hamster mAb, 9AB2, that blocks Ly-6A.2-dependent cell-cell adhesion. The 9AB2 Ab recognizes a 66-kDa glycoprotein with unique tissue expression. The 9AB2 mAb does not bind Ly-6A.2, but coimmunoprecipitates Ly-6A.2 molecule. Moreover, 9AB2 Ag-expressing thymocytes specifically bind to Chinese hamster ovary cells overexpressing Ly-6A.2 protein, and this binding is specifically blocked by 9AB2 and anti-Ly-6A.2 Abs. These results suggest that the 66-kDa protein recognized by 9AB2 mAb is the putative ligand for Ly-6A.2.

The effect of selective contracting on hospital costs and revenues.

OBJECTIVE: To examine the effects of selective contracting on California hospital costs and revenues over the 1983-1997 period. DATA SOURCES/STUDY SETTING: Annual disclosure data and discharge data sets for 421 California general acute care hospitals from 1980 to 1997. ANALYSIS: Using measures of competition developed from patient-level discharge data, and financial and utilization measures from the disclosure data, we estimated a fixed effect multivariate regression model of hospital costs and revenues. FINDINGS: We found that hospitals in more competitive areas had a substantially lower rate of increase in both costs and revenues over this extended period of time. For-profit hospitals lowered their costs and revenues after selective contracting was initiated relative to the cost and revenue levels of not-for-profit hospitals. The Medicare PPS has also led high-cost hospitals to lower their costs. CONCLUSIONS: The more competitive the hospital's market, the greater degree to which it has had to lower the rate of increase in costs. A similar pattern exists with regard to hospital revenues. Both of these trends appear to result from the growth of selective contracting. It remains unclear to what extent these cost reductions were the result of increased efficiency or of reduced quality. Since hospital cost growth is sensitive to the competitiveness of its market, antitrust enforcement is a critical element in any cost containment policy.

Can cost shifting continue in a price competitive environment?

Both Medicare and Medicaid are reducing payments to hospitals, and there is widespread concern that hospitals may respond by increasing prices to privately insured patients. Theoretical models of hospital behaviour have ambiguous predictions as to whether, and under what circumstances, hospitals will shift costs to private payers. This paper extends previous theoretical models and then tests empirically using data from California for the 1983-1991 period, a time of increasingly intense price competition. Hospitals did increase their prices to private payers in response to reductions in Medicare rates; they had far smaller and generally insignificant responses to changes in Medicaid reimbursement. Hospital ownership and the competitiveness of the hospital market both affected this behaviour, but there was no significant change over time. The results suggest the need to broaden our models of hospital behaviour to 'embed' them in their local markets.

Price competition and hospital cost growth in the United States (1989-1994).

In recent years, most health care markets in the United States (US) have experienced rapid penetration by health maintenance organizations (HMOs) and preferred provider organizations (PPOs). During this same period, the US has also experienced slowing health care costs. Using a national database, we demonstrate that HMOs and PPOs have significantly restrained cost growth among hospitals located in competitive hospital markets, but not so in the case of hospitals located in relatively concentrated markets. In relative terms, we estimate that HMOs have contained cost growth more effectively than PPOs.

CD4+ T cells mature in the absence of MHC class I and class II expression in Ly-6A.2 transgenic mice.

The TCRs expressed on T lymphocytes recognize foreign peptides bound to MHC molecules. This reactivity is the basis of specific immune response to the foreign Ag. How such specificities are generated in the thymus is still being debated. Signals generated through TCR upon interaction with self MHC-peptide complexes are critical for maturation of the CD4+ helper and CD8+ cytotoxic subsets. We have observed maturation of CD4+ but not CD8+ T cells in Ly-6A.2 transgenic MHC null mice. Since there can be no interactions with MHC molecules in these mice, these CD4+ cells must express the T cell repertoire that exists before positive and negative selection. Interestingly, despite an absence of selection by MHC molecules, the CD4+ cells that mature recognize MHC molecules at a frequency as high as in CD4+ cells in normal mice. These results demonstrate that: 1) the germline sequences encoding TCRs are biased toward reactivity to MHC molecules; and 2) CD4+ cells as opposed to CD8+ cells have distinct lineage commitment signals. These results also suggest that signals originating from Ly-6 can promote or substitute for signals generated from TCR that are required for positive selection. Moreover, this animal model offers a system to study T cell development in the thymus that can provide insights into mechanisms of lineage commitment in developing T cells.

A profile of uncompensated hospital care, 1983-1995.

This DataWatch examines national trends in the provision of uncompensated hospital care. It shows that rapid growth from 1983-1986 was followed by modest growth through 1990, a time during which managed care was becoming established in some regions. There was then another spurt in uncompensated care from 1991-1993, a period that corresponds to sizable increases in disproportionate-share payments. Uncompensated care growth again slowed through 1995. The increase in uncompensated care levels after 1988 appears not to have kept pace with growth in hospital expenses or the number of uninsured. However, the trend data do not suggest a large-scale reduction of effort.

Regulated expression of Ly-6A.2 is important for T cell development.

Ly-6A.2 is a surface protein on T cells that may play a role in lymphocyte activation. The regulation of Ly-6A.2 expression during T cell lymphopoiesis has been intriguing. It is one of the earliest markers expressed on pluripotent hemopoietic stem cells and is present on both primitive and mature T cells, but its expression is extinguished in the thymus during key developmental stages. To determine whether Ly-6A.2 is active on developing T cells, as well as the significance of its developmental regulation, Ly-6A.2 was expressed throughout T cell development under control of the T cell-specific human CD2 enhancer in transgenic mice. The constitutive overexpression of Ly-6A.2 in vivo led to a marked impairment in the generation of thymocytes. Development was arrested at the time in thymic development when Ly-6A.2 expression is normally turned off. These results indicate that the regulated expression of Ly-6A.2 in thymocytes may be important for normal development. Moreover, these findings demonstrate that Ly-6A.2 is active in the thymic microenvironment.

Overexpressed Ly-6A.2 mediates cell-cell adhesion by binding a ligand expressed on lymphoid cells.

The Ly-6 locus encodes several cell surface proteins whose functions are unknown. Although it is hypothesized that these proteins may be receptors, there is no direct evidence that they bind a ligand. Herein we present evidence that Ly-6A.2, a Ly-6 protein expressed on T lymphocytes, binds a ligand expressed on normal thymocytes and splenic B and T cells. We find that transgenic thymocytes that overexpress Ly-6A.2 spontaneously aggregate in culture. This homotypic adhesion requires the overexpression of Ly-6A.2 because it is not observed in cultures of nontransgenic thymocytes. The aggregation of Ly-6A.2 transgenic thymocytes is inhibited by phosphatidylinositol-specific phospholipase C (which removes Ly-6A.2 and other glycosylphosphatidylinositol-anchored proteins from the membrane). Some anti-Ly-6A.2 monoclonal antibodies, including nonactivating ones and Fab' fragments, inhibit this aggregation. In contrast, other anti-Ly-6A.2 monoclonal antibodies increase the aggregation of transgenic but not nontransgenic thymocytes. To further examine whether Ly-6A.2 mediates adhesion (versus inducing another adhesion pathway) reaggregation assays were performed with paraformaldehyde-fixed Tg+ thymocytes. Paraformaldehyde-fixed Tg+ thymocytes reaggregate in culture and this aggregation is also blocked by phosphatidyl-inositol-specific phospholipase C and anti-Ly-6A.2 monoclonal antibodies. These results indicate that the homotypic adhesion of cultured Ly-6A.2 transgenic thymocytes is directly mediated by Ly-6A.2 and, more importantly, strongly suggests that Ly-6A.2 binds a ligand that is expressed on thymocytes. Tg+ thymocytes also bind to nontransgenic thymocytes, B cells, and T cells, indicating that normal cells naturally express the Ly-6A.2 ligand.

Uncompensated care: hospitals' responses to fiscal pressures.

This Data Watch examines the impact of hospital competition, the Medicare prospective payment system (PPS), and Medi-Cal selective contracting on the provision of uncompensated care by private hospitals in California during 1980-1989. It finds that hospitals subject to more intense competition and greater fiscal pressure from Medicare and Medi-Cal reduced their provision of uncompensated care relative to hospitals facing less pressure from these sources. We estimate that had hospitals not been subjected to increasing price competition from growth of managed care plans and financial tightening in public programs, they would have provided 36 percent more uncompensated care than was actually provided in 1989.

A role of Ly-6A.2 in T lymphocyte activation and development.

Ly-6A.2 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed on the plasma membrane of T lymphocytes. The precise function of this molecule is not known. A role of Ly-6A.2 in T cell signaling is discussed.

A role of Ly-6A.2 in T lymphocyte activation and development

Ly-6A.2 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed on the plasma membrane of T lymphocytes. The precise function of this molecule is not known. A role of Ly-6A.2 in T cell signaling is discussed

Costs and price competition in California hospitals, 1980-1990.

Critics of health care reform proposals that incorporate managed competition contend that it has never been broadly implemented. However, insurance plans that combine insurance with the provision of care have been widely implemented and have been tested most extensively in California. This DataWatch explores California's experience with health maintenance organizations (HMOs) and preferred provider organizations (PPOs), the introduction of which was followed by overall reductions in hospital costs. These reductions were larger in competitive markets. If implemented on a national scale, such selective contracting could be expected to reduce the growth of hospital costs even more rapidly than occurred in California.

The effects of market structure and bargaining position on hospital prices.

PPOs and HMOs have gained widespread acceptance due in part to the belief that excess capacity and competitive market conditions can be leveraged to negotiate lower prices with health care providers. We investigated prices obtained in different types of markets by the largest PPO in California. Our findings indicate that greater hospital competition leads to lower prices. Furthermore, as the importance of a hospital to the PPO in an area increases, the price rises substantially. Our testing of alternative methods for defining hospital geographic markets reveals that the common practice of using counties to define the market leads to an underestimate of the price-increasing effects of a merger.

Internalization of glycosyl-phosphatidylinositol (GPI)-anchored lymphocyte proteins. II. GPI-anchored and transmembrane molecules internalize through distinct pathways.

Ly-6A.2 (T cell-activating protein, TAP) and Thy-1 are glycosyl-phosphatidyl-inositol (GPI)-anchored proteins expressed on the surface of murine T lymphocytes. We have found that Ly-6A.2 (TAP) and Thy-1 are internalized by T cells. In the present study we have investigated whether these GPI-anchored proteins enter cells by endocytosis through coated pits. Two lines of evidence argue against the involvement of coated pits in the internalization of Ly-6A.2 (TAP) and Thy-1. First, drugs that effectively blocked the endocytosis of transferrin receptor and H-2 class I molecules, (which are known to be internalized via coated pits) did not inhibit the internalization of the GPI-anchored proteins. Second, in ultrastructural analyses, Ly-6A2 (TAP) and Thy-1, in contrast to the transferrin receptor, were rarely found in coated pits or vesicles. These observations suggest that the GPI-anchored proteins on T lymphocytes are internalized by a distinct pathway that does not involve endocytosis through coated pits.

Effect of ras-activation on the expression of glycosyl-phosphatidylinositol-anchored proteins on the plasma membrane.

NIH3T3 cells were transfected with a vector containing c-H-ras under the transcriptional control of the mouse metallothionein-I promoter. When c-H-ras expression was induced with heavy metal ions there was a marked reduction in the expression of two glycosylphosphatidylinositol (GPI) anchored proteins, TAP/Ly-6A.2 and Thy-1, on the plasma membrane of the transformed cells. In contrast the cell-surface expression of other non-GPI-anchored proteins was unaltered. The major loss of Thy-1 induced by ras activation occurred from the pool of molecules expressed on the cell surface. The Thy-1 molecules that were preferentially lost from the surface of the ras-transformed cells could not be recovered from the extracellular fluid by immunoprecipitation. In contrast, the rate of internalization of Thy-1 was increased approximately 69% subsequent to ras activation. The possible significance of these findings to the function of c-H-ras is discussed.

Using DRGs to pay for inpatient substance abuse services: an assessment of the CHAMPUS reimbursement system.

In October 1988, the Civilian Health and Medical Program of the Uniformed Services (CHAMPUS) introduced a prospective payment system based on diagnostic-related groups (DRGs) to pay for substance abuse services. These services were initially excluded from the new payment system because of concerns that a DRG-based system may have a large and poorly understood financial impact on individual hospitals. This report assesses the performance of a DRG system in explaining variation in costs at the individual patient level and evaluates how well this payment system predicts resource use across hospitals. Overall, the substance abuse DRGs explained only 4.2% of the total variance in charges. It was found that the Medicare DRG-based system had to be modified to reflect the characteristics of the younger CHAMPUS population by splitting DRG 435 to account for the increased costliness of beneficiaries younger than 21 years. In addition, the study revealed substantial variation in the impact of the DRG system on hospital revenue. These differences largely reflected significant differences between general and specialty hospitals.

Internalization of phosphatidylinositol-anchored lymphocyte proteins. I. Documentation and potential significance for T cell stimulation.

Several proteins that are anchored to the surface of T lymphocytes via a phosphatidylinositol (PI) moiety can initiate cell stimulation upon cross-linking. Inasmuch as these proteins do not traverse the plasma membrane, it is not clear how they are capable of signaling across the membrane. Herein we report two distinct sets of experiments that examine the consequence of cross-linking PI-anchored molecules on murine T cells. We first analyzed the fate of antibody cross-linked TAP (Ly-6A.2) and Thy-1 molecules on T-T hybrids. Using an assay to measure receptor-mediated endocytosis, an intracellular accumulation of 125I labeled anti-TAP and anti-Thy-1 mAb was documented that was specific and Ag dependent. The internalization of these molecules was confirmed by cytotoxicity assays using antibody-toxin conjugates, and electron microscopic studies. Although the PI-anchored proteins lack a cytoplasmic domain that is necessary for the internalization of many receptors, they nevertheless can be induced to enter the cell upon cross-linking. The rate of entry of cross-linked TAP and Thy-1 into cells was shown to be 10 and 2% per hour, respectively, which is considerably less than that observed for the transferrin receptor or TCR/CD3 complex. To assess whether the internalization of TAP and Thy-1 might be of importance in their ability to stimulate T cells, we attempted to cross-link these molecules under conditions where the mAb or its cross-linked complex can not enter the cell. We observed that anti-TAP and anti-Thy-1 mAb conjugated to a cell impermeant matrix fail to stimulate T cells. This loss of stimulatory activity was observed with multiple T-T hybridomas and mAb over a wide titration of antibody concentration and was independent of the mAb isotype. Results from experiments with anti-Ig cross-linking of the mAb-PI anchored protein complex suggested that the loss of T cell stimulation upon mAb immobilization is not simply due to an alteration in the degree of antibody cross-linking. These findings were generalized to three distinct PI-anchored proteins: TAP, Thy-1, and Ly6C on normal T cells. When the same cells were stimulated through the TCR/CD3 complex, only immobilized mAb are stimulatory. These results demonstrate a marked difference in the cross-linking requirements for stimulating T cells through PI-anchored molecules in contrast to the transmembrane TCR complex. Furthermore, these findings raise the possibility that molecular internalization of Ab-PI-anchored complexes may be necessary in signaling through these molecules.