外周血嗜酸粒细胞释放的O_2~-的检测

为探讨外周血嗜酸粒细胞在IL 3、TNF α、IFN γ、佛波酯 (PMA )等刺激剂诱导下释放超氧离子 (O2 )的能力 ,我们采用超氧化物歧化酶 (SOD ) 抑制细胞色素C还原法检测嗜酸粒细胞产生O2 的量 ,结果发现 :(1)一定浓度的IL 3、PMA、TNF α能诱导嗜酸粒细胞释放大量的O2 ,与空白对照有显著性差异 ,而且以PMA的诱导能力最强 ;(2 )IFN γ的刺激量高达 10 0 0u/ml时 ,也不能有效促进嗜酸粒细胞释放O2 ,与空白对照无显著性差异 ;(3)健康献血者与哮喘等变应性疾病患者的外周血嗜酸粒细胞在相同的刺激剂诱导下释放的O2 量无显著性差异。以上结果说明 :IL 3、PMA和TNF α是嗜酸粒细胞产生O2 的强有力的诱导剂 ,而IFN γ则无此作用 ,从而提示应用IL 3、PMA和TNF α的拮抗剂有可能抑制嗜酸粒细胞释放O2 。     

Regulation of differentiation, proliferation and drug-induced apoptosis in HT58 lymphoma cells

Recently, it has been suggested, that differentiated cells are more resistant to the apoptotic effect of DNA damaging agents possibly due to the decreased activity of “damage detecting/apoptosis triggering” mechanism. Previously, we have shown, that PMA pretreatment reduced etoposide-(ETO) but enhanced staurosporine- (STA) -induced apoptosis in HT58 cells. Data presented here show that the HT58 human, “mature” B-lymphoma cells exposed to PMA secrete more IgM into the supernatant indicating commitment of cells to perform differentiated function. The sensitivity of HT58 cells to ETO- or STA-induced apoptosis is influenced diversely with PMA pre- or posttreatment. Interestingly, the DNA damage (gamma radiation, bleomycin, ETO) or okadaic acic (30 nM) reduced the [PMA+STA] - induced apoptosis.     

Application of flow cytometry and PMA-qPCR to distinguish between membrane intact and membrane compromised bacteria cells in an aquatic milieu

The paper compares two methods of distinguishing between alive and dead cells by differentiation on the basis of their membrane structure: LIVE/DEAD flow cytometry and PMA-qPCR. LIVE/DEAD flow cytometry was established using the LIVE/DEAD^® BacLight™ Bacterial Viability Kit with different ratios of Legionella pneumophila and Escherichia coli cells with intact and compromised membranes (heat treated). The PMA-qPCR method was tested and modified, and results were compared with those from LIVE/DEAD flow cytometry using L. pneumophila cells.Ratios of membrane intact to membrane compromised cells were well shown by LIVE/DEAD flow cytometry in all combinations. PMA-qPCR seems to work best in even mixed ratios (1:1) of intact and compromised cells. In other respects, we noticed an overestimation of intact cells in the samples which contained a high percentage of membrane compromised cells, and an underestimation of intact cells in samples with a small percentage of membrane compromised cells. However, looking at total counts instead of ratios, the results were within an order of magnitude. This implies that the use of PMA-qPCR is appropriate only for a qualitative analysis to monitor the success of a process such as disinfection. Furthermore, we were able to assess that both methods have advantages and disadvantages: LIVE/DEAD flow cytometry as applied in this study works well on some bacteria monocultures, but does not distinguish between bacteria species. The PMA-qPCR method allows the possibility of distinguishing between membrane intact cells and membrane compromised cells and can be used to screen for specific bacteria.     

A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by gamma-interferon

We describe a simple method by which the insoluble blue formazan dye produced by the reduction of nitro blue tetrazolium can be dissolved without heating using potassium hydroxide and dimethyl sulphoxide. This modification enhances the sensitivity and increases the applications of tests performed using the microELISA method and removes variations caused by uneven cell monolayers. It also allows quantification of NBT reduced by cells adherent to coverslips or in larger wells or Petri dishes, and can be used as a sensitive assay for macrophage activation by gamma-interferon.     

Calcium and protein kinase C enhance parathyroid hormone- and forskolin-stimulated adenylate cyclase in ROS 17/2.8 cells

Summary Both parathyroid hormone (PTH)- and forskolin-stimulated adenylate cyclase activities in ROS 17/2.8 cells are enhanced by increasing the medium concentrations of CaCl₂ from 10⁻⁵ M to 3 ×10⁻³ M. The ED₅₀ for CaCl₂ for both PTH- and forskolin-stimulated activities are similar. The tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, also enhanced both PTH- and forskolin-stimulated adenylate cyclase. This action of PMA is specific for protein kinase C as phorbol esters that are not activators of protein kinase C had no effect on the system. The combined effects of PMA and CaCl₂ were more than additive. The separate and combined effects of PMA and CaCl₂ changed the rate of activation of the enzyme (V_max) but did not modify the ED₅₀ for PTH or for forskolin. PMA and CaCl₂ both enhanced the potentiating effect of submaximal dose of forskolin on PTH-stimulated adenylate cyclase. It is concluded that calcium and PMA enhance PTH-sensitive adenylate cyclase and increase the production of cAMP by a mechanism that appears to involve the catalytic subunit of the enzyme and probably its interaction with a guanine nucleotide regulatory protein.     

Comparison of membrane-associated proteins of murine cytolytic and helper cloned T-cell lines: identification of a protein, p24, prominent in membrane fractions from cytolytic but not helper T-cells

It has recently been reported that liposomes containing membrane components from cytolytic T-cell (TC) clones could transfer lytic activity to noncytolytic T- and B-cell lines, strongly suggesting that TC possess membrane-associated molecules which noncytolytic lymphocytes lack and which play a critical role in the lytic mechanism. It was thus of interest to compare the membrane-associated proteins from TC-lines to those of noncytolytic helper T-cell (TH) lines to determine whether any membrane-associated proteins unique to TC could be identified. Cells from three TC-lines and four TH-lines were internally labelled with [35S]methionine and then disrupted by hypotonic lysis. Low-density (plasma membrane enriched) and high-density (endoplasmic reticulum enriched) membrane fractions were isolated from each cloned cell line and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Two proteins were identified which were prominent in the membrane fractions from each of the three TC-lines but not in the membrane fractions from any of the four TH-lines. One of these, p215, migrated as a broad band with an apparent mol. wt of 215,000. The other, p24, migrated as a sharp band, or tightly spaced doublet, with an apparent mol. wt of 24,000. Immunoprecipitation studies using monoclonal antibodies to T200, LFA-1, Thy 1 and Lyt 2 suggested that p215 was a variant of T200 found on TC-lines but not on TH-lines. Treatment of solubilized membrane proteins from TH-lines with anti-T200 precipitated a 185-kD protein seen on each of the TH-lines but on none of the TC-lines. In contrast, p24 was not precipitated by any of these monoclonal antibodies. It therefore appears that p24 represents a previously unidentified protein which is strongly expressed by TC but not by TH and is thus deserving of further study as to its functional significance.     

流式细胞术分析PMA诱导THP-1分化为巨噬细胞的表型特征

目的用流式细胞术明确单独使用PMA诱导THP-1细胞分化而来的巨噬细胞的分化情况。方法首先用PMA刺激THP-1细胞分化为巨噬细胞,再分别使用LPS、IFN-γ和IL-6将细胞诱导为M1型巨噬细胞,用IL-4、IL-13和IL-6将细胞诱导为M2型巨噬细胞。显微镜下观察三种细胞的形态学改变,并用流式细胞术检测各细胞主要CD分子表达的差异。结果当THP-1分化为巨噬细胞后变为贴壁生长,细胞形态呈较规则的圆形或椭圆形;M1和M2细胞形态不规则,细胞突起较明显,细胞颗粒度较大。巨噬细胞表面与抗原提呈功能相关的CD分子的表达均较低,大部分呈阴性;而M1和M2细胞表面这些CD分子的表达均较高。结论单独使用PMA刺激THP-1细胞所分化而来的巨噬细胞抗原提呈能力较弱,基本处于静息状态。     

Is subvalvular repair worthwhile in severe ischemic mitral regurgitation? Subanalysis of the Papillary Muscle Approximation trial

Objective

The symmetry of mitral valve tethering and regional left ventricle wall dysfunction are reported to play a fundamental role in the outcomes and long-term durability of surgical repair in ischemic mitral regurgitation (IMR). We recently demonstrated in a randomized clinical trial (the Papillary Muscle Approximation trial) the superiority of papillary muscle approximation (PMA) in combination with standard restrictive annuloplasty (RA) in severe IMR over annuloplasty alone in terms of adverse left ventricular remodeling and mitral regurgitation (MR) recurrence. This approach, however, failed to produce a survival advantage and was still plagued by a high incidence of reoperation. We therefore performed a subanalysis of the PMA trial on the basis of preoperative parameters to elucidate the value of subvalvular surgery in certain subcategories of patients with the aim of creating a decisional algorithm on the best operative strategy.