大鼠骨髓基质干细胞定向分化为成骨细胞及体外增殖过程中地塞米松的抑制性干预

目的:地塞米松在体外诱导骨髓基质干细胞向成骨细胞分化过程中起着关键性作用。验证骨髓基质干细胞向成骨细胞分化的能力,观察成骨细胞分化早期地塞米松对骨髓基质干细胞体外增殖的抑制效果。方法:实验于2006-09/12在南方医科大学组织工程研究中心完成。①实验方法:取5周龄雄性SD大鼠10只,经颈椎脱位法处死后取股骨,去除双侧干骺端,用DMEM高糖完全培养基冲洗骨髓腔,收集骨髓细胞,离心后按(1～2)×107L-1密度接种,加入条件培养液(DMEM高糖完全培养基,体积分数为0.1的标准胎牛血清,50mg/L维生素C,10mmol/L的B-甘油磷酸钠,100U/mL青霉素、100U/mL链霉素)进行体外培养。分别于细胞传代培养后第0,2,4天向培养基中加入1μmol/L地塞米松1mL,并设立仅加入等量培养基的空白对照组。②实验评估:以2d为间隔,倒置显微镜下观察细胞生长情况。采用CellTiter96试剂盒各组细胞增殖情况。结果:①骨髓基质干细胞向成骨细胞的分化:原代培养中贴壁细胞多呈长梭形,少数呈小圆形或三角形。原代培养六七天后进行传代,多数细胞在加入地塞米松后逐渐呈均一的长梭形,随着时间延长呈叠形多层排列,细胞外基质明显增多,并逐渐形成多个小结样结构。空白对照组细胞形态欠均一,少数细胞呈多边形或三角形,细胞外基质明显少于地寒米松组,罕见小结样结构。传代后10～12d可达80%～90%致密层,细胞生长速度较原代细胞明显增快,至第10代细胞仍未出现衰老现象。②骨髓基质干细胞的增殖检测:与空白对照组比较,细胞传代培养后第0,2,4天加入地塞米松,干预处理8,10,12d时的细胞数量均明显下降(t=5.0445～11.3795,P均<0.01)。结论:①传代的骨髓基质干细胞经地塞米松处理后,细胞形态趋于成熟,生长速度加快,可定向分化为成骨细胞。②在向成骨细胞分化早期,地塞米松能够抑制骨髓基质干细胞的体外增殖。     

Impact of prenatal diagnosis on choledochal cysts and the benefits of early excision

Aim:   To evaluate the clinical outcomes of patients with prenatally diagnosed choledochal cysts compared with those diagnosed after birth and the optimal timing of definitive treatment.
Methods:   Retrospective review of all patients who underwent primary choledochal cyst excision and Roux-en-Y hepaticojejunostomy from 1996 to 2006 at a single institution.
Results:   A total of 45 patients were included. Ten (22.2%) of the patients had prior prenatal diagnosis. The mean age at operation for this group was 4.4 months and mean follow up was 55.9 months. There was no operative complication or late morbidity. For the post-natal diagnosis group, the mean age at operation was 5.7 years ( P  < 0.000) and mean follow up period was 69 months. The most common presentation in this group was abdominal pain (31.4%), followed by pancreatitis (28.6%) and symptoms of cholestasis (25.7%). Early post-operative morbidities occurred in two (5.7%) patients. On long-term follow up, two (5.7%) further patients in the post-natal group developed complications.
Conclusion:   Prenatal diagnosis of choledochal cysts results in earlier definitive surgery. More adverse complications were seen in those who had surgery at an older age. We therefore recommend early excision of choledochal cysts.     

Offline adaptive radiotherapy for bladder cancer using cone beam computed tomography

We investigated if an adaptive radiotherapy approach based on cone beam CT (CBCT) acquired during radical treatment was feasible and resulted in improved dosimetric outcomes for bladder cancer patients compared to conventional planning and treatment protocol. A secondary aim was to compare a conventional plan with a theoretical online process where positioning is based on soft tissue position on a daily basis and treatment plan choice is based on bladder size. A conventional treatment plan was derived from a planning CT scan in the radical radiotherapy of five patients with muscle invasive bladder cancer. In this offline adaptive protocol using CBCT, the patients had 10 CBCT: daily CBCT for the first five fractions and then CBCT scan on a weekly basis. The first five daily CBCT in each patient were used to create a single adaptive plan for treatment from fraction eight onwards. A different process using the planning CT and the first five daily CBCT was used to create small, average and large bladder volumes, giving rise to small, average and large adaptive bladder treatment plans, respectively. In a retrospective analysis using the CBCT scans, we compared the clinical target volume (CTV) coverage using three protocols: (i) conventional; (ii) offline adaptive; and (iii) online adaptive with choice of ‘plan of the day’. Daily CBCT prolonged treatment time by an average of 7 min. Two of the five patients demonstrated such variation in CTV that an offline adaptive plan was used for treatment after the first five CBCT. Comparing the offline adaptive plan with the conventional plan, the CTV coverage improved from a minimum of 60.1 to 94.7% in subsequent weekly CBCT. Using the CBCT data, modelling an online adaptive protocol showed that coverage of the CTV by the 95% prescribed dose line by small, medium and large adaptive plans were 34.9, 67.4 and 90.7% of occasions, respectively. More normal tissue was irradiated using a conventional CTV to planning target volume margin (1.5 cm) compared to an online adaptive process (0.5 cm). An offline adaptive strategy improves dose coverage in certain patients to the CTV and results in a higher conformity index compared to conventional planning. Further research in online adaptive radiation therapy for bladder cancer is indicated.     

How I treat my inflammatory bowel disease-patients with thiopurines?

Thiopurines are essential drugs to maintain remission in patients with inflammatory bowel disease(IBD).Thiopurines used in IBD are azathioprine(2.0-2.5 mg/kg),mercaptopurine(1.0-1.5 mg/kg) and thioguanine(0.2-0.3 mg/kg).However,mainly due to numerous adverse events associated with thiopurine use,almost 50% of the patients have to discontinue conventional thiopurine treatment.Extensive monitoring and the application of several treatment strategies,such as split-dose administration,co-administration with allopurinol or dose reduction/increase,may increase the chance of successful therapy.With this review,we provide practical information on how thiopurines are initiated and maintained in two thiopurine research centers in The Netherlands.We provide clinical information concerning safety issues,indications and management of therapy that may serve as a guide for the administration of thiopurines in IBD patients in daily practice.     

Identification of optimal contemporary antiemetic prophylaxis for doxorubicin-cyclophosphamide chemotherapy in Chinese cancer patients: post-hoc analysis of 3 prospective studies

Objective:Chemotherapy-induced nausea and vomiting (CINV) are common with doxorubicin-cyclophosphamide (AC) chemotherapy. Recommended antiemetic regimens incorporate neurokinin-1 receptor antagonist (NK1RA), 5-hydroxytryptamine type-3 receptor antagonist (5HT3RA), corticosteroid, and dopamine antagonists. This post-hoc analysis compared results of 3 prospective antiemetic studies conducted among Chinese breast cancer patients who received (neo)adjuvant AC, in order to identify optimal antiemetic prophylaxis.Methods:A total of 304 patients were included: Group 1, ondansetron/dexamethasone (D1); Group 2, aprepitant/ondansetron/dexamethasone (D1); Group 3, aprepitant/ondansetron/dexamethasone (D1–3); Group 4, aprepitant/ondansetron/dexamethasone (D1–3)/olanzapine; and Group 5, netupitant/palonosetron/dexamethasone (D1–3). Antiemetic efficacies of Groups 3, 4, and 5 during cycle 1 of AC were individually compared with Group 1. In addition, emesis outcomes of patients in Groups 3 and 5, and those of Groups 2 and 3, were compared.Results:When comparing efficacies of a historical doublet (5HT3RA/dexamethasone) with triplet antiemetic regimens (NK1RA/5HT3RA/dexamethasone) with/without olanzapine, complete response (CR) percentages and quality of life (QOL) in overall phase of cycle 1 AC were compared between Group 1 and the other groups: Group 1 vs. 3, 41.9% vs. 38.3% (P = 0.6849); Group 1 vs. 4, 41.9% vs. 65.0% (P = 0.0107); and Group 1 vs. 5, 41.9% vs. 60.0% (P = 0.0460). Groups 4 and 5 achieved a better QOL. When comparing netupitant-based (Group 3) with aprepitant-based (Group 5) triplet antiemetics, CR percentages were 38.3% vs. 60.0%, respectively (P = 0.0176); Group 5 achieved a better QOL. When comparing 1 day (Group 2) vs. 3 day (Group 3) dexamethasone, CR percentages were 46.8% and 38.3%, respectively (P = 0.3459); Group 3 had a worse QOL.Conclusions:Aprepitant-containing triplets were non-superior to doublet antiemetics. Netupitant-containing triplets and adding olanzapine to aprepitant-containing triplets were superior to doublets. Netupitant/palonosetron/dexamethasone was superior to aprepitant/ondansetron/dexamethasone. Protracted administration of dexamethasone provided limited additional benefit.     

The hypolipidemic action of bezafibrate therapy in hypertriglyceridemia is mediated by upregulation of lipoprotein lipase: no effects on VLDL substrate affinity to lipolysis or LDL receptor binding

Fibrates are regarded as drugs of choice in hypertriglyceridemia (HTG). Downregulation of apolipoprotein (apo) C-III gene expression and upregulation of lipoprotein lipase (LPL) gene expression have been suggested to explain the hypolipidemic action of fibrates. This study was designed to study the effects of bezafibrate therapy on very low density lipoprotein (VLDL) susceptibility to lipolysis, VLDL binding to the low density lipoprotein (LDL) receptor and postheparin LPL activities in patients with HTG. VLDL lipolysis was studied with heparan sulfate proteoglycan-bound LPL. Binding affinity of VLDL to the LDL receptor was determined in J774 cells with 125I-labeled control LDL. Eighteen HTG patients were randomized to receive, in a double-blind placebo-controlled cross-over fashion, 400 mg bezafibrate once daily for 6 weeks. In response to bezafibrate therapy, plasma triglyceride and apoC-III levels decreased by 69 and 42%, respectively. HTG VLDL was lipolyzed less efficiently compared to control VLDL, and lipolysis did not improve by bezafibrate therapy. VLDL binding affinity to the LDL receptor was comparable between the control group and HTG group, and did not change upon bezafibrate therapy. However, the post-heparin LPL activity in the HTG patients increased from 153 to 192 U/l (P = 0.025). A strong inverse relation was observed between the change in LPL activities and the change in triglyceride levels (r = -0.62, P = 0.006). In conclusion, the hypolipidemic action of bezafibrate therapy in HTG may be attributed to increased LPL activity, whereas VLDL susceptibility to lipolysis and LDL receptor binding are not affected.     

Immunolocalization of notch signaling protein molecules in a maxillary chondrosarcoma and its recurrent tumor

Background

Notch receptors are critical determinants of cell fate in a variety of organisms. Notch signaling is involved in the chondrogenic specification of neural crest cells. Aberrant Notch activity has been implicated in numerous human diseases including cancers; however its role in chondrogenic tumors has not been clarified.

Method

Tissue samples from a case of primary chondrosarcoma of the maxilla and its recurrent tumor were examined immunohistochemically for Notch1-4 and their ligands (Jagged1, Jagged2 and Delta1) expression.

Results

Both primary and recurrent tumors were histopathologically diagnosed as conventional hyaline chondrosarcoma (WHO Grade I). Hypercellular tumor areas strongly expressed Notch3 and Jagged1 in spindle and pleomorphic cells suggesting up-regulation of these protein molecules at sites of tumor proliferation. Expression patterns were distinct with some overlap. Differentiated malignant and atypical chondrocytes demonstrated variable expression levels of Jagged1, and weak to absent staining for Notch1, 4 and Delta1. Protein immunolocalization was largely membranous and cytoplasmic, sometimes outlining the lacunae of malignant chondrocytes. Hyaline cartilage demonstrated a diffuse or granular precipitation of Jagged1 suggesting presence of soluble Jagged1 activity at sites of abnormal chondrogenesis. No immunoreactivity for the other Notch members was observed. Calcified cartilage was consistently Notch-negative indicating down-regulation of Notch with cartilage maturation. Stromal components namely endothelial cells and fibroblasts variably expressed Notch1, 3 and Jagged1 but were mildly or non-reactive for the other members.

Conclusions

Results indicate that Notch signaling pathway may participate in cellular differentiation and proliferation in chondrosarcoma. Findings implicate Notch3 and Jagged1 as key molecules that influence the differentiation and maturation of cells of chondrogenic lineage.     

Multiparametric analysis of colorectal cancer immune responses

Colorectal cancer(CRC) is a heterogeneous disease, with a diverse and plastic immune cell infiltrate. These immune cells play an important role in regulating tumour growth-progression or elimination. Some populations of cells have a strong correlation with disease-free survival, making them useful prognostic markers. In particular, the infiltrate of CD3~+ and CD8~+ T cells into CRC tumours has been validated worldwide as a valuable indicator of patient prognosis. However, the heterogeneity of the immune response, both between patients with tumours of different molecular subtypes, and within the tumour itself, necessitates the use of multiparametric analysis in the investigation of tumour-specific immune responses. This review will outline the multiparametric analysis techniques that have been developed and applied to studying the role of immune cells in the tumour, with a focus on colorectal cancer. Because much of the data in this disease relates to T cell subsets and heterogeneity, we have used T cell populations as examples throughout. Flow and mass cytometry give a detailed representation of the cells within the tumour in a single-cell suspension on a per-cell basis. Imaging technologies, such as imaging mass cytometry, are used to investigate increasing numbers of markers whilst retaining the spatial and structural information of the tumour section and the infiltrating immune cells. Together, the analyses of multiple immune parameters can provide valuable information to guide clinical decision-making in CRC.