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Muscle atrophy alone is insufficient to explain the significant decline in contractile force of skeletal muscle during normal aging. One contributing factor to decreased contractile force in aging skeletal muscle could be compromised excitation-contraction (E-C) coupling, without sufficient available Ca(2+) to allow for repetitive muscle contractility, skeletal muscles naturally become weaker. Using biophysical approaches, we previously showed that store-operated Ca(2+) entry (SOCE) is compromised in aged skeletal muscle but not in young ones. While important, a missing component from previous studies is whether or not SOCE function correlates with contractile function during aging. Here we test the contribution of extracellular Ca(2+) to contractile function of skeletal muscle during aging. First, we demonstrate graded coupling between SR Ca(2+) release channel-mediated Ca(2+) release and activation of SOCE. Inhibition of SOCE produced significant reduction of contractile force in young skeletal muscle, particularly at high frequency stimulation, and such effects were completely absent in aged skeletal muscle. Our data indicate that SOCE contributes to the normal physiological contractile response of young healthy skeletal muscle and that defective extracellular Ca(2+) entry through SOCE contributes to the reduced contractile force characteristic of aged skeletal muscle.

In aged skeletal muscle, changes to the composition and function of the contractile machinery cannot fully explain the observed decrease in the specific force produced by the contractile machinery that characterizes muscle weakness during aging. Since modification in extracellular Ca(2+) entry in aged nonexcitable and excitable cells has been recently identified, we evaluated the functional status of store-operated Ca(2+) entry (SOCE) in aged mouse skeletal muscle. Using Mn(2+) quenching of Fura-2 fluorescence and confocal-microscopic imaging of Ca(2+) movement from the transverse tubules, we determined that SOCE was severely compromised in muscle fibers isolated from aged mice (26-27 months) as compared with those from young (2-5 months) mice. While reduced SOCE in aged skeletal muscle does not appear to result from altered expression levels of STIM1 or reduced expression of mRNA for Orai, this reduction in SOCE is mirrored in fibers isolated from young mice null for mitsugumin-29, a synaptophysin-related protein that displays decreased expression in aged skeletal muscle. Our data suggest that decreased mitsugumin-29 expression and reduced SOCE may contribute to the diminished intracellular Ca(2+) homeostatic capacity generally associated with muscle aging.

We determined the impact of HER2 signaling on two proangiogenic factors, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), and on an antiangiogenic factor, thrombospondin-1 (TSP-1). Re-expression of HER2 in MCF-7 and T-47D breast cancer cells that endogenously express low levels of HER2 resulted in elevated expression of VEGF and IL-8 and decreased expression of TSP-1. Inhibition of HER2 with a humanized anti-HER2 antibody (trastuzumab, or Herceptin) or a retrovirus-mediated small interfering RNA against HER2 (siHER2) decreased VEGF and IL-8 expression, but increased TSP-1 expression in BT474 breast cancer cells that express high levels of HER2. These in vitro results were further evaluated by treatment of BT474 xenografts in immunosuppressed mice with trastuzumab. Trastuzumab inhibited growth of BT474 xenografts and decreased microvascular density associated with downregulation of VEGF and IL-8 and with upregulation of TSP-1 expression. Inhibiting the PI3K-AKT pathway decreased VEGF and IL-8 expression. AKT1 overexpession increased VEGF and IL-8 expression, but did not increase TSP-1 expression. A p38 kinase inhibitor, SB203580, instead blocked TSP-1 expression and a p38 activator, MKK6, increased TSP-1 expression. Trastuzumab stimulated sustained p38 activation and SB203580 attenuated the TSP-1 upregulation induced by trastuzumab. HER2 signaling therefore influences the equilibrium between pro- and antiangiogenic factors via distinct signaling pathways. Trastuzumab inhibits angiogenesis and tumor growth, at least in part, through activation of the HER2-p38-TSP-1 pathway and inhibition of the HER2-PI3K-AKT-VEGF/IL-8 pathway.

We have previously reported that breast cancer cells which overexpress HER2 produce higher levels of VEGF than cells with low levels of HER2. This study tested the hypothesis that dual targeting of the VEGF (with VEGF-Trap) and HER2 (with trastuzumab) pathways would result in greater growth inhibition of HER2-overexpressing breast cancer xenografts than either agent alone. In this study we found that human and murine endothelial cells expressed high levels of VEGF receptors (VEGFR1, VEGFR2, & VEGFR3). VEGF-Trap decreased levels of secreted VEGF derived from both human and murine cells and effectively blocked VEGF-induced tyrosine phosphorylation of VEGFR2. VEGF-Trap as a single treatment inhibited tumor microvessel density (MVD), tumor vasculature, cell proliferation, and tumor growth of BT474 xenografts in a dose-dependent manner from 2.5 mg/kg to 25 mg/kg. VEGF-Trap decreased levels of both human VEGF and PlGF protein in vivo. Trastuzumab as a single agent effectively inhibited BT474 tumor growth in a dose-dependent manner, associated with a decrease in human VEGF, tumor MVD and tumor cell proliferation. Treatment with a combination of VEGF-Trap (2.5-10 mg/kg) and trastuzumab (1 mg/kg) produced significantly greater inhibition of BT474tumor growth than either individual agent, associated with greater inhibition of tumor MVD and tumor cell proliferation. Thus, VEGF-Trap in combination with trastuzumab produces superior growth inhibition of tumor xenografts which overexpress HER2, which may result from inhibition of both tumor angiogenesis and proliferation. Similar mechanisms may contribute to the clinical anti-tumor activity of trastuzumab in combination with inhibitors of VEGF signaling pathway in women with breast cancers which overexpress HER2.

Mortality rates for coronary heart disease (CHD) have declined markedly since the early 1970s. However, CHD remains the number one cause of death in the United States. The decline in mortality has been attributed to declines in CHD risk factors (tobacco use, hypertension) and the increase in protective behaviors (exercise, weight control). Medical interventions may have also contributed to the decline in mortality. Despite these declines in mortality, racial disparities persist between Blacks and Whites. The purpose of this study was to examine the differences in receipt of cardiac catheterization, percutaneous transluminal coronary angioplasty, and coronary artery bypass graft.Data from the National Hospital Discharge Survey were used for the analysis. Patients who were Black or White andor = 40 years of age were included. Independent variables included age at discharge, sex, race, and insurance coverage. Multivariate logistic regression was used to derive odds ratios for the receipt of the three procedures by age group, sex, insurance type, and race.Significant differences (P.05) in the odds of receipt of all of cardiac catheterization, percutaneous transluminal coronary angioplasty, and coronary artery bypass graft were found by age group, insurance type, sex, and race. While the disparities persisted from 1979 to 2004, the magnitude of the differences decreased during this time period.Disparities by race, sex, and insurance type existed in the receipt of three cardiac procedures. Although differences are narrowing over time, further in-depth studies are needed to elucidate the patient, physician, and healthcare system factors associated with the disparity in receipt of these beneficial procedures.

Dynamic membrane repair and remodelling is an elemental process that maintains cell integrity and mediates efficient cellular function. Here we report that MG53, a muscle-specific tripartite motif family protein (TRIM72), is a component of the sarcolemmal membrane-repair machinery. MG53 interacts with phosphatidylserine to associate with intracellular vesicles that traffic to and fuse with sarcolemmal membranes. Mice null for MG53 show progressive myopathy and reduced exercise capability, associated with defective membrane-repair capacity. Injury of the sarcolemmal membrane leads to entry of the extracellular oxidative environment and MG53 oligomerization, resulting in recruitment of MG53-containing vesicles to the injury site. After vesicle translocation, entry of extracellular Ca(2+) facilitates vesicle fusion to reseal the membrane. Our data indicate that intracellular vesicle translocation and Ca(2+)-dependent membrane fusion are distinct steps involved in the repair of membrane damage and that MG53 may initiate the assembly of the membrane repair machinery in an oxidation-dependent manner.

A 2-tier system that classifies ovarian serous carcinoma (OSC) as low grade or high grade is gaining acceptance. Women with low-grade OSC generally have higher 5-year survival rates than do women with high-grade OSC. We examined the expression of various markers to further understand the molecular differences between low-grade and high-grade OSCs: the potential therapeutic targets or prognostic markers Her-2/neu, estrogen receptor, and progesterone receptor (PR); the metastasis-associated markers cyclin D1 (BCL1), E-cadherin, matrix metalloproteinase (MMP) 2, and MMP-9; and the cell proliferation-associated markers BCL1, Ki-67 antigen (Ki-67), and p53. For this immunohistochemical analysis, we used paraffin-embedded specimens from 47 patients with advanced-stage low-grade OSC and from 49 patients with advanced-stage high-grade OSC. Our results showed that low-grade tumors expressed significantly higher levels of estrogen receptor, PR, and E-cadherin than did high-grade tumors, suggesting the involvement of gonadal steroid hormones, especially in the pathogenesis of low-grade OSC; the PR positivity was also observed in the stromal component of these low-grade tumors. On the other hand, high-grade tumors trended toward increased expression of MMP-9, BCL1, p53, and Ki-67, and robust MMP-9 positivity was observed in the stromal component of these high-grade tumors. These differences may lead to the development of different therapeutic strategies for women with either the low-grade or the high-grade form of OSC.

Foxp3+ Regulatory T cells (Treg) suppress immune activation and play a critical role in the maintenance of self-tolerance. A subpopulation (70-80%) of Treg cells in both mouse and man express the transcription factor, Helios, but the role of Helios in Treg function is still unknown. Deletion of Helios expression in all T cells (CD4-Cre) or in early progenitors (Vav-Cre) did not result in immune dysfunction. As some studies have suggested that Helios might be expressed in activated T conventional cells, we generated mice with a conditional deletion (KO) of Helios in Treg. The selective deletion of Helios in Tregs led to a slowly progressive, systemic immune activation characterized by a Th1 profile, lymphadenopathy, splenomegaly, increased serum Ig and enhanced lymphoid follicle and germinal center formation in the absence of immune pathology. In vitro suppressive function of Helios KO Treg was normal, but KO Treg failed to differentiate into T follicular regulatory cells in vivo. While the absolute number of Treg in KO mice was enhanced, in competitive settings Helios deficient Tregs were at a striking disadvantage in the periphery. Notably, Helios KO Tregs with an activated (CD44hiCD62Llo) effector Treg phenotype were greatly decreased. Analysis of Helios KO Tregs revealed increased levels of several BH3 domain pro-apoptotic genes. These findings suggest that Helios plays a critical role in the development and/or survival of effector Tregs by repressing pro-apoptotic genes.

We identified the BRK tyrosine kinase in a PCR-based screen of tyrosine kinases expressed by ovarian tumors. BRK expression is restricted to normal differentiating epithelial cells and its overexpression may play a role in processes related to tumor development and growth. Its expression in normal ovary and ovarian tumors has not previously been described, and is the focus of this study.BRK expression levels were determined in 14 normal ovaries and 138 high-grade, late stage serous carcinomas of the ovary by immunohistochemical analysis, and in 19 ovarian cancer cell lines and immortalized ovarian surface epithelium by Western blot analysis. Furthermore, BRK/PTK6 gene copy number was determined in seven primary serous carcinomas by fluorescence in situ hybridization.Immunohistochemical studies indicate that BRK is highly expressed in 97/138 (70%) of high-grade, serous carcinomas of the ovary, but is absent in normal ovarian surface epithelia. BRK is also expressed by 9/19 of ovarian cancer cell lines, but is undetectable in immortalized ovarian surface epithelium. Interestingly, the BRK gene has been mapped to chromosome 20q13.3, a site frequently amplified in ovarian cancers, and associated with poor prognosis. We have determined by fluorescence in situ hybridization (FISH) that BRK is specifically amplified at low levels in 6/7 primary ovarian carcinomas.The amplification of the BRK gene and overexpression of BRK protein in the majority of high-grade serous carcinomas and ovarian cancer cell lines suggest that BRK may play a role in the development and growth of ovarian tumors.

Overexpression of cdc25B, an important cell cycle regulator, has been shown to result in mammary gland hyperplasia in transgenic mice and to increase steroid hormone responsiveness as a direct coactivator of the estrogen receptor (ER). We investigated the potential role of cdc25B in the pathogenesis of endometrial carcinomas in conjunction with ER-alpha. We examined the expression of cdc25B and phosphorylated ER-alpha in 4 archived human specimens of normal endometrium; 7 endometrial hyperplasia with or without atypia; 32 endometrioid endometrial carcinoma (EEC), including 20 low-grade (grade 1) and 12 high-grade (grade 2 or 3) tumors; and 18 endometrial cancers with aggressive histological subtypes (uterine papillary serous carcinoma and clear cell carcinoma, UPSC/CCC) by immunohistochemistry with monoclonal antibodies. Expression of cdc25B and phosphorylated ER-alpha was increased in endometrial hyperplasia and atypical hyperplasia compared with normal secretory endometrium. Ninety percent (18 of 20) of the low-grade EEC expressed cdc25B at a high level, whereas only 42% (5 of 12) of the high-grade EEC did so (chi(2) = 8.7; P0.01). Sixty-five percent (13 of 20) of the low-grade EEC expressed phosphorylated ER-alpha at high levels, but only 17% (2 of 12) of high-grade EEC did so (chi(2) = 7.0; P0.01). Coordinate high-level expression of phosphorylated ER-alpha and cdc25B occurred in 65% (13 of 20) of low-grade EEC but in only 17% (2 of 12) of the high-grade EEC (chi(2) = 7.0; P0.01). In the UPSC/CCC tumors, only 22% (4 of 18) of the tumors expressed phosphorylated ER-alpha at high-levels. However, 83% (15 of 18) of these carcinomas showed high expression of cdc25B (chi(2) = 13.5; P0.01). The majority of the UPSC/CCC (15 of 18) did not show coordinate high expression of phosphorylated ER-alpha and cdc25B. Our findings show that in endometrial hyperplasia and low-grade EEC, coordinate increase in cdc25B and phosphorylated ER-alpha occurs. However, in UPSC/CCC, cdc25B is highly expressed without coordinate increase in phosphorylated ER-alpha. Cdc25B may play important roles in the development and progression of EEC and UPSC/CCC by different mechanisms.

BACKGROUND Tyrosine kinases, such as c-KIT, c-ABL, and platelet-derived growth factor-beta (PDGFR-β), are important regulators of cell growth. Highly potent and selective inhibitors of tyrosine kinases are being investigated as alternatives to standard chemotherapy. One such inhibitor, imatinib mesylate, is being used to treat gastrointestinal stromal tumors and chronic myelogenous leukemia. Ovarian carcinomas frequently develop resistance to conventional chemotherapeutic agents. Immunohistochemical expression of c-ABL, PDGFR-β, and c-KIT was evaluated in ovarian carcinomas to determine whether treatment with imatinib mesylate might be feasible. METHODS The expression of c-ABL, c-KIT, and PDGFR-β in tumors was evaluated by immunohistochemical analysis of 52 ovarian serous carcinomas, including 21 low-grade (well differentiated) and 31 high-grade (poorly differentiated) tumors. Fourteen normal ovaries were also evaluated. RESULTS In normal ovarian surface epithelium, c-ABL was expressed universally. PDGFR-β was expressed in the majority (93%) of samples of normal ovarian epithelium, whereas the c-KIT protein was undetectable in normal ovarian surface epithelium. Overall, c-ABL was expressed in 71% of serous carcinomas. c-ABL was expressed more frequently in the low-grade serous carcinomas (81%) compared with the high-grade serous carcinomas (65%). PDGFR-β expression was observed in 81% of serous carcinomas overall and was observed more frequently in higher-grade tumors. c-KIT immunohistochemical staining was absent in low-grade tumors but was present in 26% of high-grade serous carcinomas. CONCLUSIONS The majority of ovarian serous carcinomas express one or more of the kinases targeted by the tyrosine kinase inhibitor, imatinib mesylate, suggesting the potential usefulness of this drug in the treatment of ovarian carcinoma. Cancer 2003;98:758–64. © 2003 American Cancer Society. DOI 10.1002/cncr.11561

Previous studies have demonstrated that activated Tregs express latent TGF-β1 on their cell surface bound to the tethering molecule GARP. Although integrins have been implicated in mediating the release of biologically active TGF-β1 from the complex of latent TGF-β1 and latent TGF-β1 binding protein, their role in processing latent TGF-β1 from the latent TGF-β1/GARP complex is unclear. We detected integrin β8 expression by CD4+Foxp3+ Treg, but not by CD4+Foxp3- T cells, by qRT-PCR. Furthermore, integrin β8 expression appeared to be a marker of thymically-derived Treg (tTreg), as its could not be detected on Foxp3+ T cells induced in vitro nor in vivo on peripherally induced Foxp3+Helios- Tregs. While Tregs from integrin β8 conditional knockouts suppressed T effector cell proliferation normally in vitro and in vivo in a model of colitis, integrin β8 deficient Tregs failed to provide a source of TGF-β1 to drive Th17 or iTreg differentiation in vitro. In addition, the knockout Tregs expressed higher levels of latent TGF-β1 on their cell surface consistent with defective processing. Taken together, integrin β8 is not only a marker of tTregs, but also functions in a cell intrinsic manner as the major pathway for processing latent TGF-β1 from the latent TGF-β1/GARP complex on the surface tTregs.

A residency training program developed by a college of pharmacy in conjunction with a home care company is described. The 12-month program is based on the ASHP Residency Learning System and the goal statements and educational objectives of ASHP's accreditation standard for pharmacy practice residency training with emphasis in home care. Establishing the program involved identifying goals, objectives, and learning experiences consistent with the expected outcomes. Specific objectives for meeting goals in four categories--practice foundation skills, direct patient care, drug information and drug policy development, and practice management--were linked to expected program outcomes. Learning experiences that would lead to achievement of the program objectives and outcomes were selected and organized into one- to eight-week rotations (e.g., in acute care, care of pediatric patients, pain management, nutrition, patient education and counseling, and administration and practice management). Throughout the program, residents gain experience in pharmaceutical services and research. Skills in care planning and monitoring are emphasized, as is practicing pharmaceutical care in an interdisciplinary environment. Residents who have completed the program have found employment immediately as pharmacy managers of home infusion centers. Pharmacy residency training in home care provides the experience needed to function as a competent clinician and manager who can identify and solve problems to improve patient care.

RATIONALE: FOXP3+ regulatory T cells (Tregs) are central to the maintenance of self-tolerance and the prevention of autoimmunity. Since they are generated both in the thymus and the periphery, the ability to distinguish these two populations would be critical to understand their development, particularly in autoimmune and inflammatory conditions. METHODS: Peripheral blood of healthy adult donors and patients with systemic lupus erythematosus as well as cord blood were analyzed for FOXP3 and Helios expression within the CD4+ T cell population by flow cytometry. Cytokine secretion and suppressive function were studied in the Tregs and correlated with Helios expression. RESULTS: Helios is expressed selectively in the FOXP3+ Tregs and is decreased with age. Helios identifies thymic-derived, cytokine non-producing FOXP3+ Tregs and not peripheral or TGFbeta-induced FOXP3+ T cells. Knockdown of Helios with siRNA does not alter the cytokine profile and suppressor function of Tregs, suggesting that Helios is not critical for their anergic and suppressive properties. CONCLUSIONS: Helios is expressed uniquely in human FOXP3+ Tregs and distinguishes the thymic over the peripheral derived Tregs. While it is expressed predominately in the Tregs, it does not appear to play a critical role in their anergic and suppressive functions.

5039 Background: Aurora-A kinase is a serine-threonine kinase involved in centrosome regulation that is frequently amplified in cancer. Its role in ovarian carcinoma has not been fully characterize...

Junctophilin (JP) mediates the close contact between cell surface and intracellular membranes in muscle cells ensuring efficient excitation-contraction coupling. Here we demonstrate that disruption of triad junction structure formed by the transverse tubular (TT) invagination of plasma membrane and terminal cisternae of sarcoplasmic reticulum (SR) by reduction of JP expression leads to defective Ca2+ homeostasis in muscle cells. Using adenovirus with small hairpin interference RNA (shRNA) against both JP1 and JP2 genes, we could achieve acute suppression of JPs in skeletal muscle fibers. The shRNA-treated muscles exhibit deformed triad junctions and reduced store-operated Ca2+ entry (SOCE), which is likely due to uncoupled retrograde signaling from SR to TT. Knockdown of JP also causes a reduction in SR Ca2+ storage and altered caffeine-induced Ca2+ release, suggesting an orthograde regulation of the TT membrane on the SR Ca2+ release machinery. Our data demonstrate that JPs play an important role in controlling overall intracellular Ca2+ homeostasis in muscle cells. We speculate that altered expression of JPs may underlie some of the phenotypic changes associated with certain muscle diseases and aging.