Human LY6 gene family: potential tumor-associated antigens and biomarkers of prognosis in uterine corpus endometrial carcinoma.

The human Lymphocyte antigen-6 (LY6) gene family has recently gained interest for its possible role in tumor progression. We have carried out in silico analyses of all known LY6 gene expression and amplification in different cancers using TNMplot and cBioportal. We also have analyzed patient survival by Kaplan-Meier plotter after mining the TCGA database. We report that upregulated expression of many LY6 genes is associated with poor survival in uterine corpus endometrial carcinoma (UCEC) cancer patients. Importantly, the expression of several LY6 genes is elevated in UCEC when compared to the expression in normal uterine tissue. For example, LY6K expression is 8.25× higher in UCEC compared to normal uterine tissue, and this high expression is associated with poor survival with a hazard ratio of 2.42 (p-value = 0.0032). Therefore, some LY6 gene products may serve as tumor-associated antigens in UCEC, biomarkers for UCEC detection, and possibly targets for directing UCEC patient therapy. Further analysis of tumor-specific expression of LY6 gene family members and LY6-triggered signaling pathways is needed to uncover the function of LY6 proteins and their ability to endow tumor survival and poor prognosis in UCEC patients.

Ly-6A-Induced Growth Inhibition and Cell Death in a Transformed CD4+ T Cell Line: Role of Tumor Necrosis Factor-α.

Ly-6A, a member of the Ly-6/uPAR supergene family of proteins, is a cell adhesion and cell signaling protein. Signaling through Ly-6A activates the cell-intrinsic apoptotic cell death pathway in CD4+ T cell lines, as indicated by the release of cytochrome C, and activation of caspases 9 and 3. In addition, Ly-6A induces cytokine production and growth inhibition. The mechanism underlying the distinct cellular responses that are triggered by engaging Ly-6A protein has remained unknown. To examine the relatedness of these distinct responses, we have quantified the production of pro-apoptotic, growth inhibitory and tumor suppressive cytokines, such as TNF-α, TGF-ß and a related protein GDF-10, in response to Ly-6A signaling. Anti-Ly-6A monoclonal antibody-induced activation of YH16.33 CD4+ T cell line generated low levels of TGF-ß and GDF-10 but elevated levels of TNF-α. Blocking the biological activity of TNF-α resulted in reduced Ly-6A-induced apoptosis in T cells. The Ly-6A-induced response in the T cell line was distinct, as signaling through the antigen receptor complex did not cause growth inhibition and apoptosis despite high levels of TGF-ß and GDF-10 that were detected in these cultures. Additionally, in response to antigen receptor complex signaling, lower amount of TNF-α was detected. These results indicate the contribution of TNF-α in the observed Ly-6A-induced growth inhibition and apoptosis and provide a mechanistic explanation for the biologically distinct responses observed in CD4+ T cells after engaging Ly-6A protein. Additionally, the findings reported here will aid in the understanding of inhibitory signaling initiated by Ly-6A protein, especially in the context of its potential immune checkpoint inhibitory role in T cells.

Cholesterol-dependent plasma membrane order (Lo) is critical for antigen-specific clonal expansion of CD4+ T cells.

Early "T cell activation" events are initiated within the lipid microenvironment of the plasma membrane. Role of lipid membrane order (Lo) in spatiotemporal signaling through the antigen receptor in T cells is posited but remains unclear. We have examined the role of membrane order (Lo)/disorder (Ld) in antigen specific CD4+ T cell activation and clonal expansion by first creating membrane disorder, and then reconstituting membrane order by inserting cholesterol into the disordered plasma membrane. Significant revival of antigen specific CD4+ T cell proliferative response was observed after reconstituting the disrupted membrane order with cholesterol. These reconstitution experiments illustrate Koch's postulate by demonstrating that cholesterol-dependent membrane order (Lo) is critical for responses generated by CD4+ T cells and point to the importance of membrane order and lipid microenvironment in signaling through T cell membrane antigen receptors.

Nonviral gene delivery to T cells with Lipofectamine LTX.

Retroviral gene delivery is widely used in T cell therapies for hematological cancers. However, viral vectors are expensive to manufacture, integrate genes in semirandom patterns, and their transduction efficiency varies between patients. In this study, several nonviral gene delivery vehicles, promoters, and additional variables were compared to optimize nonviral transgene delivery and expression in both Jurkat and primary T cells. Transfection of Jurkat cells was maximized to a high efficiency (63.0% ± 10.9% EGFP+  cells) by transfecting cells with Lipofectamine LTX in X-VIVO 15 media. However, the same method yielded a much lower transfection efficiency in primary T cells (8.1% ± 0.8% EGFP+ ). Subsequent confocal microscopy revealed that a majority of the lipoplexes did not enter the primary T cells, which might be due to relatively low expression levels of heparan sulfate proteoglycans detected via messenger RNA-sequencing. Pyrin and HIN (PYHIN) DNA sensors (e.g., AIM2 and IFI16) that can induce apoptosis or repress transcription after binding cytoplasmic DNA were also detected at high levels in primary T cells. Therefore, transfection of primary T cells appears to be limited at the level of cellular uptake or DNA sensing in the cytoplasm. Both of these factors should be considered in the development of future viral and nonviral T cell gene delivery methods.

Engaging Ly-6A/Sca-1 triggers lipid raft-dependent and -independent responses in CD4+ T-cell lines.

INTRODUCTION: The lymphocyte antigen 6 (Ly-6) supergene family encodes proteins of 12-14 kda in molecular mass that are either secreted or anchored to the plasma membrane through a glycosyl-phosphatidylinisotol (GPI) lipid anchor at the carboxy-terminus. The lipidated GPI-anchor allows localization of Ly-6 proteins to the 10-100 nm cholesterol-rich nano-domains on the membrane, also known as lipid rafts. Ly-6A/Sca-1, a member of Ly-6 gene family is known to transduce signals despite the absence of transmembrane and cytoplasmic domains. It is hypothesized that the localization of Ly-6A/Sca-1 with in lipid rafts allows this protein to transduce signals to the cell interior. METHODS AND RESULTS: In this study, we found that cross-linking mouse Ly-6A/Sca-1 protein with a monoclonal antibody results in functionally distinct responses that occur simultaneously. Ly-6A/Sca-1 triggered a cell stimulatory response as gauged by cytokine production with a concurrent inhibitory response as indicated by growth inhibition and apoptosis. While production of interleukin 2 (IL-2) cytokine by CD4+ T cell line in response to cross-linking Ly-6A/Sca-1 was dependent on the integrity of lipid rafts, the observed cell death occurred independently of it. Growth inhibited CD4+ T cells showed up-regulated expression of the inhibitory cell cycle protein p27kip but not of p53. In addition, Ly-6A/Sca-1 induced translocation of cytochrome C to the cytoplasm along with activated caspase 3 and caspase 9, thereby suggesting an intrinsic apoptotic cell death mechanism. CONCLUSIONS: We conclude that opposing responses with differential dependence on the integrity of lipid rafts are triggered by engaging Ly-6A/Sca-1 protein on the membrane of transformed CD4+ T cells.

Investigating B Cell Development, Natural and Primary Antibody Responses in Ly-6A/Sca-1 Deficient Mice.

Ly-6A/Stem cell antigen-1 (Ly-6A/Sca-1) is a glycosylphosphatidylinositol-anchored protein expressed on many cell types including hematopoietic stem cells (HSCs) and early lymphoid-specific progenitors. Ly-6A/Sca-1 is expressed on CD4+ T cells and plays a role in regulating cellular responses to foreign antigens. The role of Ly-6A/Sca-1 in primary antibody responses has not been defined. To investigate whether Ly-6A/Sca-1 functions in humoral immunity, we first injected Ly-6A/Sca-1-deficient and wild-type control mice with chicken ovalbumin (c-Ova) protein mixed with an adjuvant. We then assessed the ability of the mice to generate a primary antibody response against cOva. We further examined the development of B cells and circulating antibody isotypes in non-immunized Ly-6A/Sca-1deficient mice to determine if Ly6A/Sca-1 functions in development irrespective of antigen-specific immune activation. Ly-6A/Sca-1/Sca-1-deficient mice did not show any significant changes in the number of B lymphocytes in the bone marrow and peripheral lymphoid tissues. Interestingly, Ly-6A/Sca-1/Sca-1-/- mice have significantly elevated serum levels of IgA with λ light chains compared to wild type controls. B cell clusters with high reactivity to anti-IgA λ monoclonal antibody were detected in the lamina propria of the gut, though this was not observed in the bone marrow and peripheral lymphoid tissues. Despite these differences, the Ly-6A/Sca-1deficient mice generated a similar primary antibody response when compared to the wild-type mice. In summary, we conclude that the primary antibody response to cOva antigen is similar in Ly-6A/Sca-1deficient and sufficient mice. In addition, we report significantly higher expression of the immunoglobulin λ light chain by B cells in lamina propria of Ly-6A/Sca-1deficient mice when compared to the wild-type control.

Lipid raft-based membrane order is important for antigen-specific clonal expansion of CD4(+) T lymphocytes.

BACKGROUND: Lipid rafts are cholesterol and saturated lipid-rich, nanometer sized membrane domains that are hypothesized to play an important role in compartmentalization and spatiotemporal regulation of cellular signaling. Lipid rafts contribute to the plasma membrane order and to its spatial asymmetry, as well. The raft nanodomains on the surface of CD4(+) T lymphocytes coalesce during their interaction with antigen presenting cells (APCs). Sensing of foreign antigen by the antigen receptor on CD4(+) T cells occurs during these cell-cell interactions. In response to foreign antigen the CD4(+) T cells proliferate, allowing the expansion of few antigen-specific primary CD4(+) T cell clones. Proliferating CD4(+) T cells specialize in their function by undergoing differentiation into appropriate effectors tailored to mount an effective adaptive immune response against the invading pathogen. RESULTS: To investigate the role of lipid raft-based membrane order in the clonal expansion phase of primary CD4(+) T cells, we have disrupted membrane order by incorporating an oxysterol, 7-ketocholesterol (7-KC), into the plasma membrane of primary CD4(+) T cells expressing a T cell receptor specific to chicken ovalbumin323-339 peptide sequence and tested their antigen-specific response. We report that 7-KC, at concentrations that disrupt lipid rafts, significantly diminish the c-Ovalbumin323-339 peptide-specific clonal expansion of primary CD4(+) T cells. CONCLUSIONS: Our findings suggest that lipid raft-based membrane order is important for clonal expansion of CD4(+) T cells in response to a model peptide.

In vitro derivation of interferon-γ producing, IL-4 and IL-7 responsive memory-like CD4(+) T cells.

CD4(+) memory is critical for successful protection against pathogenic challenge. As such, understanding the heterogeneity of cells that arise and survive after initial stimulation of naïve CD4(+) T cells will aid in the design of more successful vaccines. In previous studies, in vivo experimental systems have been extensively used to generate functional memory responses by lymphocytes. Here, we have attempted to develop an in vitro experimental system to generate memory CD4(+) T lymphocytes. CD4(+) T cells stimulated through the antigen receptor complex were examined for their memory-like characteristics after 3 weeks of cell culture. A subset of surviving cells expressed high levels of CD44 and low levels of CD45RB (CD44(hi)CD45(lo)), a phenotype that is similar to bonafide memory CD4(+) T cells. In vitro generated memory-like CD4(+) T cells secreted higher levels of IFN-γ, with rapid kinetics, upon re-stimulation than their naïve counterparts. In addition, these memory-like CD4(+) T cells did not produce either IL-2 or IL-4 but readily proliferated when cultured in the presence of IL-7 and IL-4. These observations suggest that CD4(+) cells surviving the expansion phase of immune response produce a Th1-signature cytokine and retain responsiveness to IL-4, a Th-2 cytokine, as well as to a well described survival factor, interleukin-7.

Analysis of detergent-free lipid rafts isolated from CD4+ T cell line: interaction with antigen presenting cells promotes coalescing of lipid rafts.

BACKGROUND: Lipid rafts present on the plasma membrane play an important role in spatiotemporal regulation of cell signaling. Physical and chemical characterization of lipid raft size and assessment of their composition before, and after cell stimulation will aid in developing a clear understanding of their regulatory role in cell signaling. We have used visual and biochemical methods and approaches for examining individual and lipid raft sub-populations isolated from a mouse CD4+ T cell line in the absence of detergents. RESULTS: Detergent-free rafts were analyzed before and after their interaction with antigen presenting cells. We provide evidence that the average diameter of lipid rafts isolated from un-stimulated T cells, in the absence of detergents, is less than 100 nm. Lipid rafts on CD4+ T cell membranes coalesce to form larger structures, after interacting with antigen presenting cells even in the absence of a foreign antigen. CONCLUSIONS: Findings presented here indicate that lipid raft coalescence occurs during cellular interactions prior to sensing a foreign antigen.

The relationship between safety net activities and hospital financial performance.

BACKGROUND: During the 1990's hospitals in the U.S were faced with cost containment charges, which may have disproportionately impacted hospitals that serve poor patients. The purposes of this paper are to study the impact of safety net activities on total profit margins and operating expenditures, and to trace these relationships over the 1990s for all U.S urban hospitals, controlling for hospital and market characteristics. METHODS: The primary data source used for this analysis is the Annual Survey of Hospitals from the American Hospital Association and Medicare Hospital Cost Reports for years 1990-1999. Ordinary least square, hospital fixed effects, and two-stage least square analyses were performed for years 1990-1999. Logged total profit margin and operating expenditure were the dependent variables. The safety net activities are the socioeconomic status of the population in the hospital serving area, and Medicaid intensity. In some specifications, we also included uncompensated care burden. RESULTS: We found little evidence of negative effects of safety net activities on total margin. However, hospitals serving a low socioeconomic population had lower expenditure raising concerns for the quality of the services provided. CONCLUSIONS: Despite potentially negative policy and market changes during the 1990s, safety net activities do not appear to have imperiled the survival of hospitals. There may, however, be concerns about the long-term quality of the services for hospitals serving low socioeconomic population.

Cell-free antibody capture method for analysis of detergent-resistant membrane rafts.

Cholesterol-rich microdomains present on the plasma membrane appear to play an important role in spatio-temporal regulation of cell signaling and cell adhesion processes. Compositional heterogeneity of these microdomains and their coalescence during cell-cell interactions may provide one mechanism for triggering and/or regulating signaling cascades from the plasma membrane to the cell interior. Biochemical analyses of distinct lipid microdomain subpopulations and single-rafts obtained from unstimulated and ligand-stimulated cells are critical for deciphering functional role of lipid rafts. We have designed a cell-free assay that captures detergent-resistant lipid rafts with an antibody against a raft-resident molecule and detects the presence of another lipid raft molecule. Moreover, this cell-free assay provides a simple and quick way to examine the simultaneous presence of two proteins in the lipid rafts, and has the potential to estimate trafficking of molecules in and out of the lipid microdomains during cell signaling on a single lipid raft-basis.

Interferon gamma enhances clonal expansion and survival of CD4+ T cells.

Interferon-gamma (IFN-gamma) serves numerous functions in the regulation of the immune response. During the early phase of the immune response IFN-gamma is produced by natural killer and natural killer T cells. Although the effects of this cytokine on antigen presenting cells and other cell types are known, its direct role on CD4(+) T cells remains unclear. We demonstrate that CD4(+) T cells exposed to IFN-gamma proliferate more vigorously than the controls in response to signals through the antigen receptor. The increased proliferation of IFN-gamma-treated CD4(+) T cells is not due to enhanced signaling through the antigen receptor, but is accounted for by their increased survival. Our data suggest that enhanced survival of IFN-gamma-treated CD4(+)T cells is independent of signal transducer and activator of transcription 1 (STAT 1), a transcription factor that controls the expression of a variety of IFN-gamma-targeted genes. In addition, we demonstrate that independent of STAT 1, IFN-gamma treatment increases the expression of double-stranded RNA-dependent protein kinase, a kinase involved in regulating protein synthesis. Taken together, our findings suggest a direct role of IFN-gamma on unstimulated CD4(+) T cells that is likely to enhance the advent of adaptive immunity by augmenting their survival during the initiation of the immune response.

Marginal cost of emergency department outpatient visits: an update using California data.

OBJECTIVE: We sought to clarify the importance of time frame in the measurement of marginal cost and to provide marginal cost estimates for outpatient emergency department (ED) visits that better reflect current economic conditions. DATA SOURCES: Analyses are based upon data that California hospitals report to the Office of Statewide Health Planning and Development (OSHPD). The time period covered was 1990 through 1998. Hospitals without EDs, or hospitals designated as trauma centers, were excluded from the analysis. STUDY DESIGN: Nine years of panel data were used to estimate hospital cost functions, which were then used to test for economies of scale and to derive estimates of both short- and long-run marginal costs (excluding the physician expense component). PRINCIPAL FINDINGS: We found only weak evidence in favor of scale economies and, in that context, we argue that long-run marginal costs should be the preferred metric for judging the cost of treating outpatient ED visitors. We estimate these long-run costs (in 1998 dollars) to be roughly 348 dollars per visit for large urban hospitals, 288 dollars for other urban hospitals, 203 dollars for rural hospitals, and 314 dollars overall. CONCLUSIONS: Our results suggest that the marginal cost of an outpatient ED visit is larger than is commonly believed. A key implication of this finding is that hospital administrators need to think more carefully about their nonurgent care policies, especially as they pertain to ED operations.

A novel approach to examining compositional heterogeneity of detergent-resistant lipid rafts.

Lipid rafts play an important role in cell signalling, cell adhesion and other cellular functions. Compositional heterogeneity of lipid rafts provides one mechanism of how lipid rafts provide the spatial and temporal regulation of cell signalling and cell adhesion. The constitutive presence of some signalling receptors/molecules and accumulation of others in the lipid raft allows them to interact with each other and thereby facilitate relay of signals from the plasma membrane to the cell interior. Devising a method that can analyze these lipid microdomains for the presence of signalling receptors/molecules on an individual raft basis is required to address the issue of lipid raft heterogeneity. SDS-PAGE analysis, currently used for analyses of detergent-resistant lipid rafts, does not address this question. We have designed a cell-free assay that captures detergent-resistant lipid rafts with an antibody against a raft-resident molecule and detects the presence of another lipid raft molecule. Our results suggest that detergent-resistant lipid rafts, also known as detergent-resistant membranes, are heterogeneous populations on an immortalized mouse T-cell plasma membrane with respect to antigen receptor/signalling complex and other signalling/adhesion proteins. This cell-free assay provides a simple and quick way to examine the simultaneous presence of two proteins in the lipid rafts and has the potential to estimate trafficking of molecules in and out of the lipid microdomains during cell signalling on a single detergent-resistant lipid raft basis.

Evidence of cost shifting in California hospitals.

We used 1993-2001 data from private hospitals in California to investigate whether decreases in Medicare and Medicaid prices were associated with increases in prices paid for privately insured patients. We found that a 1 percent relative decrease in the average Medicare price is associated with a 0.17 percent increase in the corresponding price paid by privately insured patients; similarly, a 1 percent relative reduction in the average Medicaid price is associated with a 0.04 percent increase. These relationships imply that cost shifting from Medicare and Medicaid to private payers accounted for 12.3 percent of the total increase in private payers' prices from 1997 to 2001.

The cost of an emergency department visit and its relationship to emergency department volume.

STUDY OBJECTIVE: This article addresses 2 questions: (1) to what extent do emergency departments (EDs) exhibit economies of scale; and (2) to what extent do publicly available accounting data understate the marginal cost of an outpatient ED visit? Understanding the appropriate role for EDs in the overall health care system is crucially dependent on answers to these questions. The literature on these issues is sparse and somewhat dated and fails to differentiate between trauma and nontrauma hospitals. We believe a careful review of these questions is necessary because several changes (greater managed care penetration, increased price competition, cost of compliance with Emergency Medical Treatment and Active Labor Act regulations, and so on) may have significantly altered ED economics in recent years. METHODS: We use a 2-pronged approach, 1 based on descriptive analyses of publicly available accounting data and 1 based on statistical cost models estimated from a 9-year panel of hospital data, to address the above-mentioned questions. RESULTS: Neither the descriptive analyses nor the statistical models support the existence of significant scale economies. Furthermore, the marginal cost of outpatient ED visits, even without the emergency physician component, appear quite high--in 1998 dollars, US295 dollars and US412 dollars for nontrauma and trauma EDs, respectively. These statistical estimates exceed the accounting estimates of per-visit costs by a factor of roughly 2. CONCLUSION: Our findings suggest that the marginal cost of an outpatient ED visit is higher than is generally believed. Hospitals thus need to carefully review how EDs fit within their overall operations and cost structure and may need to pay special attention to policies and procedures that guide the delivery of nonurgent care through the ED.

Mouse Ly-6 proteins and their extended family: markers of cell differentiation and regulators of cell signaling.

The Ly-6 locus on mouse chromosome 15 encodes a family of 10-12 kDa proteins that are linked to the cell surface by a glycosylphosphatidyl-inositol anchor and have cell signaling and cell adhesion properties. Expression of Ly-6 proteins is tightly regulated during development; these proteins continue to serve as excellent differentiation surface markers on normal and abnormal cells, but their role in driving cellular differentiation is still emerging. Recent studies suggest that Ly-6 gene products participate in regulating signaling through other cell type-specific receptors, perhaps by virtue of these proteins being localized in lipid rafts that play a key role in relaying signals from the membrane to the nucleus. Ligands for some Ly-6 proteins have been reported; the consequence of their interactions with the Ly-6 receptor remains to be fully uncovered. Mouse Ly-6-like proteins have also been reported from a variety of life forms ranging from Caenorhabditis elegans to humans that show a limited amino acid identity and share structural features with members of mouse Ly-6. Despite these similarities, the non-murine Ly-6 proteins bind distinct ligands and appear to have different cellular functions. All members of the Ly-6 super gene family perhaps evolved from an ancestral gene by a gene duplication mechanism.

Hospital emergency departments: the authors respond.

In response to a perceived crisis in California's emergency department (ED) capacity, Glenn Melnick and colleagues sought to construct an empirical database that could bring objective data to bear on this important issue. In this response they address some of the substantive issues raised by the authors of four preceding commentaries. These issues include the use of aggregates and averages, the omission of trauma centers, staffing shortages, and overcrowding. In their view, the paper has added reliable new information to better understand the underlying economics faced by community hospitals with EDs and how they have responded over the past decade.

Emergency department capacity and access in California, 1990-2001: an economic analysis.

Media report that hospitals are closing their emergency departments (EDs) and reducing access to ED services, raising concerns that EDs are not sustainable under competition and managed care. We analyzed financial, economic, capacity, and utilization data for California EDs for 1990-2001. We found that contrary to media reports, hospitals are not abandoning the ED market. Rather, our results show a robust market, where hospitals are adding ED capacity to meet increased demand and to maintain access. Supporting economic analyses show that EDs are sustainable since they generate a sizable and growing portion of inpatient admissions, which contribute to overall economic viability.