Transfection of Lepidopteran insect cells with Baculovirus DNA

Summary Several protocols are presented for preparation and transfection of Baculovirus and plasmid DNAs into Lepidopteran insect cells using the calcium-phosphate co-precipitation technique. Important parameters for optimum efficiency include the inherent susceptibility of the recipient cell line for transfection, and the method of preparation of viral and plasmid DNAs. The protocols presented provide reproducible high efficiencies for transfection of several Lepidopteran cell lines.     

P-selectin glycoprotein ligand-1 mediates rolling of mouse bone marrow-derived mast cells on P-selectin but not efficiently on E-selectin

It has been shown recently that mast cells play an essential role as a source of tumor necrosis factor-α production during neutrophil recruitment to sites of bacterial infection. Increased numbers of mast cells are indeed noted at sites of wound healing and inflammation. These cells are either recruited from the bone marrow or proliferate locally under cytokine stimulation. Little is known about how mast cell progenitors extravasate into tissue. Using antibody-like fusion proteins of mouse E-selectin and P-selectin, we have analyzed the ability of immature mouse bone marrow-derived mast cells (BMMC) to interact with the endothelial selectins. The P-selectin glycoprotein ligand-1 (PSGL-1) was affinity-isolated from detergent extracts of surface biotinylated BMMC with both selectin-IgG fusion proteins. However, only P-selectin-IgG, but not E-selectin-IgG showed significant interaction with intact BMMC as tested by flow cytometry and cell attachment assays with the immobilized fusion proteins under flow and non-flow conditions at physiological shear stress. Thus, in spite of carrying the necessary carbohydrate modifications which enable solubilized PSGL-1 to bind avidly to E-selectin, PSGL-1 on the surface of BMMC is presented in a way that prevents it from interacting efficiently with E-selectin. Affinity-purified rabbit antibodies against mouse PSGL-1 almost completely blocked the interaction of BMMC with P-selectin-IgG in flow cytometry as well as in cell adhesion assays under static and under flow conditions. Our data reveal that PSGL-1 is the major binding site for P-selectin on mouse BMMC progenitors, but does not support efficient interactions with E-selectin.     

Human malignant mesothelial cells: Variability of ultrastructural features in established and nude mice transplanted cell lines

The aim of this study was to determine, by transmission electron microscopy, the differentiation features of 21 human malignant mesothelioma cell lines (HMCLs) established from 13 specimens of 12 confirmed human malignant mesotheliomas, and of tumours induced in nude mice injected with 16 HMCLs. Fifty per cent of HMCLs showed typical mesothelial differentiation (long and slender microvilli, desmosomes, perinuclear intermediate filaments); 29 per cent did not show differentiation; and the remainder were poorly differentiated. Three human tumour specimens gave several different HMCLs; the cell lines obtained from a given tumour exhibited variable mesothelial differentiation. Eleven HMCLs were compared with the native tumour. Four were similar to the tumour and seven were less well differentiated, in most cases in relation to their microvilli. With six HMCLs, tumours induced in nude mice were less well differentiated than the corresponding cell lines, whereas with four HMCLs, tumours were equally or better differentiated. However, in most nude mice tumours, typical mesothelial microvilli were present. These results show that cell lines established from malignant mesothelioma may exhibit dedifferentiated features. However, while the variability in ultrastructural differentiation may result from the culture microenvironment, it could also be related to the state of differentiation, of the native tumour sample and to tumour cell heterogeneity.     

Sympathetic nervous activity and cardiovascular variability after a 3-day tail suspension in rats

The effects of a 3-day tail suspension on central and peripheral sympathetic activity were studied in rats by determining the in vivo noradrenaline (NA) turnover in the brain cell groups involved in central blood pressure control (A1, A2, A5 and A6) and in two peripheral organs, heart and kidneys. In addition, cardiovascular parameters and their variabilities were investigated by recording blood pressure (BP) and heart rate (HR) before and after suspension. These measurements were processed by spectrum analysis to assess the influence of tail suspension on autonomic balance. The NA turnover in the suspended rats was markedly reduced in A2 (–49%, P<0.01) and A5 (–38%, P<0.01) nuclei but unchanged in A1 and A6 cell groups compared with the control rats. Peripheral NA turnover was decreased in cardiac atria (–44%, P<0.001) and ventricles (–27%, P<0.01) while it was unchanged in kidneys after suspension. The BP, HR and their variabilities were similar in both groups of animals and showed no changes after suspension compared with baseline values. Spectrum analysis of BP and HR in our conscious suspended rats revealed no changes in power spectrum density or in peak frequencies. The discrepancy between the decrease in central sympathetic activity and the absence of changes in cardiovascular parameters after tail suspension raises the question of the validity of the tail suspended rat model when studying the cardiovascular deconditioning observed in humans after an exposure to actual or simulated weightlessness.     

Mast cell populations in the chick embryo lung and their response to compound 48/80 and dexamethasone

Summary Two mast cell populations, connective tissue mast cells (CTMCs) and mucosal mast cells, (MMCs) containing different proteoglycans in their granules, can be distinguished in several animal species by means of histochemical methods. In this study we documented the presence of these two types of mast cell in the chick embryo lung, from the 15th incubation day for the MMCs, and from the 18th incubation day for the CTMCs. Lungs of embryos treated with compound 48/ 80, which produces degranulation of the CTMCs, showed a decrease in the number of this type of mast cell and an unchanged number of MMCs. In the lungs of embryos treated with dexamethasone, which degranulates MMCs, a reduction in the number of these cells and an unchanged number of the CTMs were found.     

Toll样受体与树突状细胞

Toll样受体(TLR)在介导固有免疫和适应性免疫应答中有重要作用,可以表达于多种免疫细胞,包括树突状细胞(DC).了解Toll样受体的免疫学基础、与DC之间的联系以及其在免疫耐受干预方面的作用很有必要.     

Postoperative spindle cell nodule of the breast: Pseudosarcomatous myofibroblastic proliferation following endo‐surgery

Despite the frequent use of fine‐needle aspiration, core biopsy and surgery, postoperative spindle cell nodule (PSCN) is a rare pathological complication that may be diagnostically treacherous. Presented herein is the case of a 52‐year‐old woman who developed a 7 mm mammary nodular lesion 66 days after removal of an area of columnar cell hyperplasia involving cellular and architectural atypia, performed with the Mammotome Breast Biopsy System. The lesion was highly cellular and composed of intersecting fascicles of plump spindle cells with blunt‐ended elongated nuclei and nucleoli easily visible. Interspersed mononuclear cells and hemosiderin‐laden macrophages were evident. PSCN is a reactive, benign myofibroblastic proliferation. Differential diagnosis includes benign and malignant spindle cell lesions of the breast. Recognition of this reactive lesion will avoid overdiagnosis of spindle cell malignant tumor. Attention to clinicopathological and histological features should result in accurate recognition of this lesion.     

离心作用测定细胞与材料间的粘附力及其初步应用

目的：介绍一种简单易行的检测细胞与材料间黏附力的方法。方法：本方法的建立基于离心力在转速一定时与半径成正比的原理。医用石膏制备培养皿固定装置，于培养皿底制备材料薄膜（包括有PLGA，PLGA COLI，PLGA COL I FN），待大鼠骨骼肌卫星细胞悬液与其作用一定时间后，一定转速开动离心机。测定未脱落细胞斑的半径R,取均值。结果：上述三种材料表面未脱落大鼠骨骼肌卫星细胞圆斑半径（R）分别为7.96、15.11、26.10mm，三组间有显著性差异。结论：（1）R的变化可反映细胞与材料间粘附力的改变。（2）利用离心作用检测细胞与材料间的粘附力是可行的，尤其适用于同一种细胞与不同材料间粘附力的比较。（3）COL I和FN的使用可以增加大鼠骨骼肌卫星细胞与PLGA间的粘附。     

Physiological and Psychological Effects of Delivering Medical News Using a Simulated Physician–Patient Scenario

We examined the acute stress response associated with having to deliver either bad or good medical news using a simulated physician–patient scenario. Twenty-five healthy medical students were randomly assigned to a bad medical news (BN), a good medical news (GN), or a control group that read magazines during the session. Self-report measures were obtained before and after the task. Blood pressure and heart rate were measured throughout the task period. Four blood samples were obtained across the task period. The BN and GN tasks produced significant increases in self-reported distress and cardiovascular responses compared with the control group. There was also a significant increase in natural killer cell function 10 min into the task in the BN group compared with the control group. The BN task was also somewhat more stressful than the GN task, as shown by the self-report and cardiovascular data. These findings suggest that a simulated physician–patient scenario produces an acute stress response in the physician, with the delivery of bad medical news more stressful than the delivery of good medical news.     

Clonality analysis of different histological components in combined small cell and non-small cell carcinoma of the lung

Combined small cell and non-small cell carcinoma is relatively rare in the lung. Examination of the clonal relationship of different components in this type of tumor may give a clue to the rarity. We retrieved 6 such tumors; all 6 had small cell carcinoma and adenocarcinoma components, and 3 had an additional squamous cell carcinoma component. We examined the point mutations in the p53 gene and allelic loss (ie, the loss of heterozygosity [LOH] pattern) of chromosome 3p in each component. p53 mutations were detected in the small cell carcinoma component of 5 tumors and in the non-small cell carcinoma components of 2 tumors. In 1 case, the squamous cell carcinoma component had a p53 mutation locus identical to that in the small cell carcinoma component, but in the other case, the adenocarcinoma component had a different mutation than that in the small cell carcinoma component. Chromosome 3p LOH loci in the squamous cell carcinoma component were present in the small cell carcinoma component in all 3 cases, but some LOH loci were not identical in the small cell carcinoma and adenocarcinoma components in 3 cases. These results suggest that the small cell and squamous cell carcinoma components of combined small cell lung carcinomas have an intimate clonal relationship. On the other hand, the adenocarcinoma component often may be derived from a separate clone or, more likely, undergo a progressive process separate from the squamous cell-small cell carcinoma beginning in a very early stage, that is, before the appearance of p53 and chromosome 3p abnormalities. This tumorigenesis process may explain the relative rarity of combined small cell and non-small cell carcinoma, which occurs primarily in the peripheral lung, an infrequent site of squamous cell carcinoma.