异源性Th表位修饰的CTLA4-Ig—msBlys构建及融合蛋白表达

目的构建异源性Th表位修饰的CTLA4-Ig-msBlys的原核表达质粒,并在EcoliBL21中诱导融合蛋白表达,初步检测蛋白特性。方法采用PCR法从质粒pCMV-sBlys扩增sBlys片段,将与卵清蛋白（ovalbumin,OVA）Th表位重组后的msBlys连接CTLA4-Ig片段,克隆入pGEX-KG原核表达载体,用LB固体培养基抗生素筛选、酶切并经琼脂糖凝胶电泳鉴定;将质粒pGEX—KG-CTI。A4-Ig—msBlys转入E．coliBL21菌株中,实现插入基因的融合表达,用SDS-PAGE和Western-blot检测表达产物。结果pGEX—KG—GTLA4-Ig—msBlys构建正确且最终转人E．coliBL21,得到融合蛋白的表达;CTLA4-Ig-msBlys融合蛋白在E．coli中获得表达,表达产物的相对分子质量同预期值一致。结论成功构建原核表达质粒pGEX-KG-CTLA4-Ig-msBlys,并在E．coliBL21表达CTLA4-Ig-msBlys融合蛋白,为下一步探讨Blys在自身免疫性疾病患者的治疗作用奠定了基础。     

Shared governance as vertical alignment of nursing group power and nurse practice council effectiveness

Aim(s)  This study validates an instrument for measuring the effectiveness of nursing practice councils and offers a framework for measuring and understanding shared governance.
Background  Empowerment results from the vertical alignment of nursing group power with nursing unit power practices. The field lacks an instrument for measuring nurses' practice of power.
Method(s)  Two studies ( n 1 = 119; n 2 = 248) are used to validate the Nursing Practice Council effectiveness scale (NPCes).
Results  NPCes is a valid and reliable index of nursing practice council effectiveness. This study suggests specific diagnostic tools to understand two levels for actualized power, one at the group or departmental level and one at the unit level.
Conclusion(s)  NPCes and the Sieloff-King Assessment of Group Power within Organizations (SKAGPO) can be used together to improve examination of shared governance. Examining group power as well as unit-level practices may give a more complete view of barriers to nurse empowerment.
Implications for nursing management  Changing nursing power and practices in an organization may be made more effective by engaging and monitoring vertical alignment of strategies fostering power competencies among nurse leaders and simultaneously supporting nursing practice councils as a means of exercising nurse authority at the unit level.     

Enzyme-linked immunoabsorbent assay detection of a soluble form of urokinase plasminogen activator receptor in vivo

The receptor for urokinase plasminogen activator (uPA-R, CD87) is a glycosylphosphatidylinositol (GPI)-anchored 50 to 65 kD glycoprotein that, by regulating membrane-associated plasmin activity, may facilitate the invasion of inflammatory and malignant cells. Certain other GPI-anchored glycoproteins are shed from the cell membrane and exist as soluble products in vitro and in vivo. To determine if uPA-R undergoes a similar phenomenon, we have developed a sensitive enzyme- linked immunoabsorbent assay (ELISA) (using a rabbit antiserum as both capture and detection reagents) to measure the quantity of soluble uPA- R (suPA-R) in tissue culture supernatants and biologic fluids. Using this ELISA, we have detected suPA-R in the culture supernatants of U- 937 cells and human monocytes stimulated in vitro by certain soluble inflammatory mediators (Sitrin et al, Blood 84:1268, 1994; Mizukami et al., Clin Res 42:115A, 1994). To determine if suPA-R exists in vivo, we have screened the plasma of 20 normal volunteers (mean +/- SD, 3 +/- 3 ng/mL; median, 2 ng/mL; range, 1 to 11 ng/mL [serum values slightly higher]); the plasma of 13 ICU patients with clinical sepsis syndrome (mean +/- SD, 30 +/- 11 ng/mL; median, 11 ng/mL; range, 4 to 221 ng/mL); and the extravascular fluids (pleural, pericardial, and peritoneal) of 84 individuals with presumed inflammatory or malignant conditions (mean +/- SD, 21 +/- 39 ng/mL; median, 10 ng/mL; range, 2 to 253 ng/mL). Among the latter specimens, most were inflammatory exudates (only six were malignant by positive cytology) with the highest quantities of suPA-R associated with neutrophilic exudates. The solubility of suPA-R contained within these fluids was confirmed by reanalysis after ultracentrifugation to remove particulate material. When tested in a uPA ligand capture ELISA, representative specimens of extravascular body fluids and sepsis plasma contained suPA-R capable of binding uPA ligand (generally representing a small fraction of the immunoreactive material). We conclude from these data that suPA-R is immunologically detectable in vitro and in vivo with high concentrations of receptor found under conditions of inflammatory stimulation. The possibility of suPA-R's biologic activity is suggested by its partial retention of ligand binding capacity.     

Efficient retrovirus transduction of mouse pluripotent hematopoietic stem cells mobilized into the peripheral blood by treatment with granulocyte colony-stimulating factor and stem cell factor

Cytokine-mobilized peripheral blood cells have been shown to participate in hematopoietic recovery after bone marrow (BM) transplantation, and are proposed to be useful targets for retrovirus- mediated gene transfer protocols. We treated mice with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) to mobilize hematopoietic progenitor cells into the peripheral blood. These cells were analyzed for the number and frequency of pluripotent hematopoietic stem cells (PHSC). We found that splenectomized animals treated for 5 days with G-CSF and SCF showed a threefold increase in the absolute number of PHSC over normal mice. The number of peripheral- blood PHSC increased 250-fold from 29 per untreated mouse to 7,200 in peripheral-blood PHSC in splenectomized animals treated for 5 days with G-CSF and SCF. Peripheral blood PHSC mobilized by treatment with G-CSF and SCF were analyzed for their ability to be transduced by retroviral vectors. Peripheral-blood PHSC from splenectomized animals G-CSF and SCF were transduced with a recombinant retrovirus containing the human MDR-1 gene. The frequency of gene transfer into peripheral blood PHSC from animals treated for 5 and 7 days was two-fold and threefold higher than gene transfer into PHSC from the BM of 5-fluorouracil-treated mice (P < .01). We conclude that peripheral blood stem cells mobilized by treatment with G-CSF and SCF are excellent targets for retrovirus- mediated gene transfer.     

Bone marrow collected 14 days after in vivo administration of granulocyte colony-stimulating factor and stem cell factor to mice has 10-fold more repopulating ability than untreated bone marrow

We have examined the repopulating ability of bone marrow and peripheral blood cells collected immediately and at intervals after treatment of donor mice with the combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF). Using a competitive repopulation assay we showed that the repopulating ability of peripheral blood cells was highest immediately after cytokine treatment and declined to normal levels within 6 weeks of the termination of treatment with G-CSF and SCF. In contrast the repopulating ability of bone marrow cells was low immediately after cytokine treatment and increased to levels that were 10-fold or more greater than marrow from untreated mice by 14 days after termination of treatment with G-CSF and SCF. This high level of repopulating activity declined to normal levels by 6 weeks after termination of treatment with G-CSF and SCF. The high level of repopulating ability was confirmed by injecting cells from G- CSF- and SCF-treated donors into unconditioned recipients. Peripheral blood cells collected immediately after treatment with G-CSF and SCF engrafted into unconditioned mice sevenfold better than an equivalent number of bone marrow cells from untreated mice. Likewise, bone marrow cells collected 14 days after treatment of the donor animal with G-CSF and SCF engrafted at 10-fold higher levels than an equivalent number of bone marrow cells from untreated mice. We conclude that the treatment of donor mice with G-CSF and SCF causes a transient increase in the repopulating ability of peripheral blood and later of bone marrow. These observations may have applications to clinical hematopoietic stem cell transplantation.     

Lysozyme enhances monocyte-mediated tumoricidal activity: a potential amplifying mechanism of tumor killing

The mononuclear phagocyte is well established as an in vitro cytotoxic effector cell for certain human tumors. The mechanism(s) for this action remains unclear. Increased levels of lysozyme, a cationic enzyme synthesized in large amounts by mononuclear phagocytes, are associated with increased resistance to transplantable animal tumors. In this study, we provide evidence that human lysozyme, isolated from the urine of leukemic patients, has marked potentiating effects on human monocyte- tumor-cell cytocidal activity. In addition, lysozyme-exposed monocytes incorporate increased quantities of leucine, suggesting that monocytes are capable of amplifying their own metabolic activation by secreting an endogenous constituent. Tri-N-acetyl-glucosamine, a competitive inhibitor for the active site of lysozyme, inhibits cytocidal activity. Conversely, protamine, an extraneous albeit similarly positively charged molecule, increases monocyte-mediated tumor cytotoxicity; this protamine effect is negated by heparin. We conclude that lysozyme, at least partially by its positive charge, is capable of enhancing in vitro monocyte tumor cell cytotoxicity; its in vivo secretion may potentiate monocyte-tumor-cell interaction.     

The Leeds Evaluation of Efficacy of Detoxification Study (LEEDS) project: An open-label pragmatic randomised control trial comparing the efficacy of differing therapeutic agents for primary care detoxification from either street heroin or methadone [ISRCTN07752728]

Background

Heroin is a synthetic opioid with an extensive illicit market leading to large numbers of people becoming addicted. Heroin users often present to community treatment services requesting detoxification and in the UK various agents are used to control symptoms of withdrawal. Dissatisfaction with methadone detoxification [8] has lead to the use of clonidine, lofexidine, buprenorphine and dihydrocodeine; however, there remains limited evaluative research. In Leeds, a city of 700,000 people in the North of England, dihydrocodeine is the detoxification agent of choice. Sublingual buprenorphine, however, is being introduced. The comparative value of these two drugs for helping people successfully and comfortably withdraw from heroin has never been compared in a randomised trial. Additionally, there is a paucity of research evaluating interventions among drug users in the primary care setting. This study seeks to address this by randomising drug users presenting in primary care to receive either dihydrocodeine or buprenorphine.

Methods/design

The Leeds Evaluation of Efficacy of Detoxification Study (LEEDS) project is a pragmatic randomised trial which will compare the open use of buprenorphine with dihydrocodeine for illicit opiate detoxification, in the UK primary care setting. The LEEDS project will involve consenting adults and will be run in specialist general practice surgeries throughout Leeds. The primary outcome will be the results of a urine opiate screening at the end of the detoxification regimen. Adverse effects and limited data to three and six months will be acquired.

     

Building confidence into communication of bad news: The role of the patient advocate

The need for a patient advocate is greater than ever as medical errors continue to occur. News media quickly capture the egregious errors, but more errors are experienced by patients who suffer quietly. These patients know something wrong occurred during their hospitalization, but they choose to refrain from pursuing litigation against the providers. There also are thousands of individuals who never realize that a medical error occurred. In a patient‐ and family‐centered care environment, patient advocates can bridge these issues by participating on the healthcare team that is involved with the initial disclosure of the event and by providing a caring relationship to assure the patient's voice is heard and understood.