建立大鼠骨髓源性树突状细胞体外扩增及纯化的方法

目的:目前对树突状细胞的研究主要集中在人及小鼠系统,关于大鼠树突状细胞特性和功能的研究较少。体外扩增并纯化大鼠骨髓树突状细胞的方法,并对其进行形态学和免疫学鉴定。方法:实验于2004-06/2005-06在第四军医大学西京医院耳鼻喉科实验室完成。实验材料:8～12周龄雌性SD大鼠由第四军医大学实验动物中心提供,实验过程中对动物处置符合动物伦理学标准。实验方法:①参考Oliver方法,利用黏附法诱导分离SD大鼠骨髓细胞,获得高纯度的树突状细胞。采用扫描电镜和透射电镜观察培养6,8,10,12 d与12 d加内毒素情况下细胞形态,并采用流式细胞仪鉴定。②无菌条件下取出大鼠脾脏加RPMI1640培养液研磨并通过不锈钢筛网,收集未贴壁细胞,以RPMI1640培养液冲洗出的细胞作为反应细胞。取96孔培养板加入反应细胞,并按照1∶480,1∶240,1∶120,1∶60比例加入刺激细胞。采用ELX800型酶联免疫检测仪测定波长为490 nm处吸光度值。结果:①随培养时间的增长,细胞逐渐成熟,培养12 d的细胞形态不规则,表面有大量树突状突起,培养12 d加内毒素的细胞进一步成熟,体积增大,高表达CD80、CD86分子。②随着培养时间和树突状细胞数目的增加,树突状细胞刺激T细胞增殖的能力逐渐增强,加入内毒素后这种能力进一步加强。结论:成功地建立了体外大量扩增大鼠骨髓树突状细胞方法,并可在培养过程中收获不同成熟状态树突状细胞。     

The role of PKA and PKCε pathways in prostaglandin E2-mediated hypernociception

Background and purpose:

Protein kinase (PK) A and the ε isoform of PKC (PKCε) are involved in the development of hypernociception (increased sensitivity to noxious or innocuous stimuli) in several animal models of acute and persistent inflammatory pain. The present study evaluated the contribution of PKA and PKCε to the development of prostaglandin E₂ (PGE₂)-induced mechanical hypernociception.

Experimental approach:

Prostaglandin E₂-induced mechanical hypernociception was assessed by constant pressure rat paw test. The activation of PKA or PKCε was evaluated by radioactive enzymic assay in the dorsal root ganglia (DRG) of sensory neurons from the hind paws.

Key results:

Hypernociception induced by PGE₂ (100 ng) by intraplantar (i.pl.) injection, was reduced by i.pl. treatment with inhibitors of PKA [A-kinase-anchoring protein St-Ht31 inhibitor peptide (AKAPI)], PKCε (PKCεI) or adenylyl cyclase. PKA activity was essential in the early phase of the induction of hypernociception, whereas PKC activity was involved in the maintenance of the later phase of hypernociception. In the DRG (L4-L5), activity of PKA increased at 30 min after injection of PGE₂ but PKC activity increased only after 180 min. Moreover, i.pl. injection of the catalytic subunit of PKA induced hypernociception which was markedly reduced by pretreatment with an inhibitor of PKCε, while the hypernociception induced by paw injection of PKCε agonist was not affected by an inhibitor of PKA (AKAPI).

Conclusions and implications:

Taken together, these findings are consistent with the suggestion that PKA activates PKCε, which is a novel mechanism of interaction between these kinases during the development of PGE₂-induced mechanical hypernociception.     

Use of Conjoint Analysis to Assess Breast Cancer Patient Preferences for Chemotherapy Side Effects

Objective.

Our objective was to evaluate preferences associated with grade I/II and grade III/IV chemotherapy side effects among breast cancer patients receiving chemotherapy. We also assessed trade-offs that patients are willing to make between treatment side effects and the route and schedule of treatment administration.

Methods.

In this cross-sectional study, patients receiving chemotherapy for breast cancer completed a one-time Web survey. Conjoint analysis was used to elicit preferences for 17 grade I/II and III/IV side effects associated with available chemotherapies and regimens. In the analysis, the risk of each side effect was increased by 5%, holding all others constant, and the respective impact on patient preferences was identified.

Results.

A total of 102 women participated (mean age 54 ± 11). Among the grade I/II side effects, a 5% reduction in the risk of sensory neuropathy, nausea, and motor neuropathy had the highest impact on preferences. Among grade III/IV side effects, motor neuropathy, nausea/vomiting, and myalgia made the most difference. An oral twice-daily regimen was most preferred; however, patients were willing to receive an intravenous regimen relative to oral to avoid an increased risk of 5% in the majority of side effects. Avoiding an increased chance of grade III/IV motor neuropathy was associated with willingness to tolerate one of the least preferred administration schedules.

Conclusion.

This study identified relative preferences among both mild/moderate to severe side effects from the patient perspective. Patients appear to be willing to make trade-offs between side effects and different regimens. These findings may help to inform medical decision-making processes.     

Minor depression and physical outcome trajectories in heart failure and pulmonary disease

We examined conjoint trajectories of depression-physical illness outcomes in elderly inpatients with minor depression and heart failure or pulmonary disease, and identified demographic, psychosocial, physical, and treatment predictors of trajectory. Consecutively admitted patients over age 50 with heart failure and/or chronic pulmonary disease were screened for minor depression using the Structured Clinical Interview for Depression. Follow-up evaluations were performed at 6 and 12 weeks using the Longitudinal Interview Follow-Up Evaluation, Hamilton Depression Scale, and Chronic Heart Failure-Chronic Respiratory Disease Questionnaire. Patients were placed into four depression-physical illness outcome trajectories: (T#1) depression better, illness better; (T#2) depression better, illness same; (T#3) depression same, illness better; and (T#4) depression same, illness same. Bivariate and multivariate predictors were examined. Minor depression was identified in 587 patients. Of these, 487 were evaluated at 6 weeks and 444 at 12 weeks. By 6 weeks, 39.4% of patients improved both on depression and physical illness (T#1), and 27.3% improved on neither (T#4). By 12 weeks, 49.6% had improved on both and 20.5% on neither. Race, admitting hospital, past psychiatric history, family psychiatric history, comorbid physical illnesses, and antidepressant drug treatment independently predicted outcome trajectory. Improvements in depression and physical illness track closely together in elderly inpatients with heart failure or pulmonary disease. Baseline patient characteristics predict which outcome trajectory they are likely to follow after hospital discharge, and may be useful in diagnosis and management.     

Use and delivery of granulocyte colony-stimulating factor in breast cancer patients receiving neoadjuvant or adjuvant chemotherapy-single-centre experience

Background

Use of granulocyte colony-stimulating factor (g-csf) as primary prophylaxis against chemotherapy-induced neutropenia has significant cost implications. We examined use of g-csf for early-stage breast cancer patients at our centre. The study also examined the pattern of nurse-led patient teaching with respect to drug self-administration.

Methods

Patients who received g-csf between November 2009 and October 2010 were identified from pharmacy records. After consent had been obtained, electronic charts were examined to extract data on chemotherapy and use of g-csf. Patients were contacted by telephone to obtain information on the utilization of home-care nursing visits for g-csf administration.

Results

The study analyzed 36 patients. Median age was 58 years (range: 31–78 years). Of the 36 patients, 30 (83%) had received adjuvant treatment, and 6 (17%), neoadjuvant treatment. Most patients (71%) received 10 days (range: 7–10 days) of filgrastim. Of the 36 patients, 29 (81%) received g-csf as primary prophylaxis. In 90% of those patients, primary prophylaxis commenced with the taxane component of treatment. Of the 36 patients, 7 (19%) received g-csf after neutropenia, including 2 who had febrile neutropenia. In 96% of the patients, injections were received at home with the help of a nurse; those patients were subsequently taught self-injection techniques. The median number of nursing visits was 2 (range: 1–3 visits). Most patients were satisfied with the home care and g-csf teaching they received.

Conclusions

Most of the g-csf used in breast cancer treatment during the study period was given for primary prophylaxis. A major reason for the decision to use g-csf appears to have been physician-perceived risk of febrile neutropenia. Delivery of g-csf by home-care nurses was well received by patients.     

Excision of the primary tumour in patients with metastatic breast cancer: a clinical dilemma

Background

Approximately 10% of new breast cancer patients will present with overt synchronous metastatic disease. The optimal local management of those patients is controversial. Several series suggest that removal of the primary tumour is associated with a survival benefit, but the retrospective nature of those studies raises considerable methodologic challenges. We evaluated our clinical experience with the management of such patients and, more specifically, the impact of surgery in patients with synchronous metastasis.

Methods

We reviewed patients with primary breast cancer and concurrent distant metastases seen at our centre between 2005 and 2007. Demographic and treatment data were collected. Study endpoints included overall survival and symptomatic local progression rates.

Results

The 111 patients identified had a median follow-up of 40 months (range: 0.6–71 months). We allocated the patients to one ot two groups: a nonsurgical group (those who did not have breast surgery, n = 63) and a surgical group (those who did have surgery, n = 48, 29 of whom had surgery before the metastatic diagnosis). When compared with patients in the nonsurgical group, patients in the surgical group were less likely to present with T4 tumours (23% vs. 35%), N3 nodal disease (8% vs. 19%), and visceral metastasis (67% vs. 73%). Patients in the surgical group experienced longer overall survival (49 months vs. 33 months, p = 0.01) and lower rates of symptomatic local progression (14% vs. 44%, p < 0.001).

Conclusions

In our study, improved overall survival and symptomatic local control were demonstrated in the surgically treated patients; however, this group had less aggressive disease at presentation. The optimal local management of patients with metastatic breast cancer remains unknown. An ongoing phase iii trial, E2108, has been designed to assess the effect of breast surgery in metastatic patients responding to first-line systemic therapy. If excision of the primary tumour is associated with a survival benefit, then the preselected subgroup of patients who have responded to initial systemic therapy is the desired population in which to put this hypothesis to the test.     

Lipoxin A4 attenuates zymosan-induced arthritis by modulating endothelin-1 and its effects

BACKGROUND AND PURPOSE

Lipoxin A₄ (LXA₄) is a lipid mediator involved in the resolution of inflammation. Increased levels of LXA₄ in synovial fluid and enhanced expression of the formyl peptide receptor 2/lipoxin A₄ receptor (FPR2/ALX) in the synovial tissues of rheumatoid arthritis patients have been reported. Endothelins (ETs) play a pivotal pro-inflammatory role in acute articular inflammatory responses. Here, we evaluated the anti-inflammatory role of LXA₄, during the acute phase of zymosan-induced arthritis, focusing on the modulation of ET-1 expression and its effects.

EXPERIMENTAL APPROACH

The anti-inflammatory effects of LXA₄, BML-111 (agonist of FPR2/ALX receptors) and acetylsalicylic acid (ASA) pre- and post-treatments were investigated in a murine model of zymosan-induced arthritis. Articular inflammation was assessed by examining knee joint oedema; neutrophil accumulation in synovial cavities; and levels of prepro-ET-1 mRNA, leukotriene (LT)B₄, tumour necrosis factor (TNF)-α and the chemokine KC/CXCL1, after stimulation. The direct effect of LXA₄ on ET-1-induced neutrophil activation and chemotaxis was evaluated by shape change and Boyden chamber assays respectively.

KEY RESULTS

LXA₄, BML-111 and ASA administered as pre- or post-treatment inhibited oedema and neutrophil influx induced by zymosan stimulation. Zymosan-induced preproET-1 mRNA, KC/CXCL1, LTB₄ and TNF-α levels were also decreased after LXA₄ pretreatment. In vitro, ET-1-induced neutrophil chemotaxis was inhibited by LXA₄ pretreatment. LXA₄ treatment also inhibited ET-1-induced oedema formation and neutrophil influx into mouse knee joints.

CONCLUSION AND IMPLICATION

LXA₄ exerted anti-inflammatory effects on articular inflammation through a mechanism that involved the inhibition of ET-1 expression and its effects.