Recirculating and germinal center B cells differentiate into cells responsive to polysaccharide antigens

Antibodies against bacterial capsular polysaccharides play a critical protective role. Responses to these antigens can occur without the help or control of T cells and are associated with marginal zone (MZ) B cells. Capsular antigens are diverse and some cross-react with self-carbohydrate epitopes. This diversity may explain the recruitment of non-autoreactive recirculating B cells and memory B cells to the MZ in addition to other B cells, some of which are weakly autoreactive cells, that are recruited to the MZ without entering the recirculating pool. To test whether memory B cells respond to polysaccharide-based antigens, mice with hapten-specific memory B cells were challenged with hapten-polysaccharide. Hapten-specific plasma cells producing high affinity antibody with Ig V-region mutations were induced. To test whether naive recirculating B cells can form MZ cells that respond to polysaccharide, recirculating B cells from lymph nodes were transferred into Rag-1-deficient mice. MZ cells differentiated from the donor cells without proliferation or T cell help and responded to polysaccharide-based antigen. The differentiation of B cells both from germinal centers and the recirculating pool to the MZ phenotype is likely to make an important contribution to the repertoire of B cells that respond to polysaccharide antigens.     

Comparison of the unlabeled and labeled pre-mRNA splicing assays in vitro

Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome that catalyzes the removal of non-coding intron sequences to ligate exons into mature mRNA prior to transport and translation. The purpose of our study is to explore whether the in vitro unlabeled pre-mRNA splicing assay could be performed as an alternative method of splicing reaction other than the radiolabeled one. Two different splicing methods in vitro , P labeled and unlabeled pre-mRNA as the substrates in the reaction, were investigated. The radiolabeled products were visualized by autoradiography while the unlabeled products were observed by Ethidium Bromide (EB) staining. As a result, although there are more unspecific bands in the EB staining assay than 32P labeled one, the RNA products of in vitro splicing could be observed clearly. This suggests that the unlabeled pre-mRNA splicing assay can be an optional substitution for the isotope-labeled assay.     

冷冻切片常遇到的问题及解决对策

目的 提高制作冷冻切片质量，确保实验结果科学性。方法 通过对切片机工作室温度设置选择，防卷板的调整应用及切片常遇到的问题分析，阐述提高制作高质量冷冻切片的方法。结果 通过对冷冻切片机有关参数的设定，提高了切片质量。结论 冷冻切片常遇到的问题，通过及时调整切片机相应设置，可以便捷解决。     

Identification of mast cells in bronchoalveolar lavage fluid

The present study was carried out to compare the effectiveness of different fixation and staining methods in the identification of mast cells obtained by bronchoalveolar lavage from patients with interstitial lung diseases. Cell preparations were fixed with formaldehyde or methanol. Mast cells were identified by metachromatic staining with May Grünwald Giemsa, Toluidine blue or Gallamine blue Giemsa. After formaldehyde fixation only a few mast cells were identified, regardless of the stain method used. In contrast, a significantly higher number of mast cells were observed after methanol fixation. Using this fixative, Toluidine blue stain showed a higher number of mast cells than May Grünwald Giemsa. However, there was no difference between Toluidine and Gallamine blue Giemsa in the number of cells observed. The easy identification of mast cells after staining with toluidine, combined with its easy application, suggest that Toluidine blue stain after methanol fixation is the most useful method for determining the presence of mast cells in lavage fluid.     

局灶性脑缺血后老年大鼠室管膜下区和颗粒下层细胞增殖与分化的实验研究

目的观察老年大鼠局灶性脑缺血后室管膜下区（SVZ）和颗粒下层（SGZ）神经干细胞的增殖与分化.方法取老年大鼠制作大脑中动脉梗塞模型.用5-溴脱氧尿核苷（BrdU）脉冲标记结合免疫组织化学单标记技术,观察正常组、假手术组、脑缺血后3、7、14、21、28 d组SVZ和SGZ区BrdU阳性细胞的变化;用BrdU累积标记结合免疫组织化学双标技术,观察脑缺血14 d后SVZ和SGZ区BrdU/NeuN和BrdU/GFAP双标阳性细胞的数量.结果在正常组、假手术组及各脑缺血组大鼠的双侧SVZ和SGZ均可观察到BrdU阳性细胞.与正常组和假手术组相比,脑缺血后SVZ和SGZ区BrdU阳性细胞明显增加.缺血组SVZ区BrdU阳性细胞在脑缺血后7 d时达到高峰,28 d时仍高于正常水平;SGZ区BrdU阳性细胞在脑缺血后14 d时达到高峰,28 d时仍高于正常水平.通过BrdU累积标记和免疫组织化学双标发现：脑缺血14 d后,老年大鼠SVZ区有部分细胞显示BrdU/NeuN（0.98%）或BrdU/GFAP（12.56%）双标阳性,而SGZ区未见双标细胞.结论局灶性脑缺血可激活老年大鼠室管膜下区和颗粒下层的神经干细胞明显增殖,并且室管膜下区有部分增殖细胞可分化为神经元或神经胶质.     

声带鳞状细胞癌早期改变的病理学观察

目的探讨声带鳞状细胞癌早期病理学的特点，提高病理诊断水平。方法总结89例声带鳞状细胞癌早期改变病例的病理资料，对其石蜡切片进行HE染色、PAS染色及p53、Ki-67免疫组化染色；以59例声带角化症（分为单纯增生组40例和异型增生组19例）和30例声带浸润癌（浸润深度〉3mm的癌）作为对照。结果在HE染色下，声带鳞状细胞癌的早期改变可区分为两种类型：Ⅰ型为上皮全层癌变型，占67．4％（60／89）；Ⅱ型为上皮基底层及副基底层癌变型，占32．6％（29／89），又可分为Ⅱa和Ⅱb两个亚型。HE染色显示有可疑微小浸润者52例，PAS染色示其中的43例（83％）的可疑病灶周边基膜样物质消失，有微浸润，Ⅰ型微浸润的比例较Ⅱ型明显偏低（P=0．007）。HE染色下3例（3．4％，3／89）认为无微浸润者经深切证实有浸润，并经PAS染色确认。Ⅰ型和Ⅱ型的p53表达率差异无显著性（P=0．445），而Ki-67阳性率Ⅰ型高于Ⅱ型（P=0．048）。癌早期改变组的p53阳性率高于声带角化症伴单纯增生组（P=0．008），而与声带角化症异型增生组和声带进展癌组之间的差异无统计学意义（P=0．240，P=0．268）。癌早期改变组的Ki-67阳性率明显低于浸润癌组（P=0．000），并明显高于角化症伴单纯增生组（P=0．001），但与角化症伴异型增生组之间差异无显著性（P=0．248）。结论声带鳞状细胞癌早期改变可区分为Ⅰ型和Ⅱ型，Ⅱ型癌变可在不累及上皮全层的情况下，由上皮的基底层和（或）副基底层细胞直接向固有膜内增生及癌变，此型占全部病例的近1／3，早期浸润是Ⅱ型诊断的可靠依据；Ⅱ型的存在提示声带鳞状细胞癌的早期发生和演进可能存在不同的机制；PAS染色和p53、Ki-67免疫组化染色有助于声带鳞状细胞癌Ⅱ型早期的诊断。     

Cell populations of the ganglion cell layer: displaced amacrine and matching amacrine cells in the pigeon retina

Summary The proportion and size distribution of ganglion and non-ganglion cells in the ganglion cell layer of different areas of the pigeon retina was examined in whole-mounts of the retina by retrograde axonal transport of horseradish peroxidase (HRP) from large brain injections. A maximum of 98% of cells were labelled in the red field and a maximum of 77% in the peripheral yellow field. Unlabelled cell bodies were 30% smaller than labelled ganglion cells and had a mean diameter of 6.2 m and a size range of 4 to 9 m. The morphology of cells in the ganglion cell layer was examined by Golgi staining of retinal whole-mounts. Small glia, displaced amacrine and ganglion cells were found. Displaced amacrine cell bodies were about 30% smaller than ganglion cells and their size distribution was similar to the unlabelled cells in HRP preparations. Displaced amacrine cells had small rounded cell bodies (mean diameter 6.2 m) increasing in size with eccentricity, and a unistratified dendritic tree of fine, nearly radial, varicose dendrites in sublamina 4 of the inner plexiform layer. They had elliptical dendritic fields (mean diameter 66 m) aligned parallel to the retina's horizontal meridian. A population of amacrine cells was found with somas at the inner margin of the inner nuclear layer and soma and dendritic morphology matching those of displaced amacrines. These amacrine cells had unistratified dendritic trees at the junction of sublaminae 1 and 2 of the inner plexiform layer. Pigeon displaced amacrine cells and their matching amacrines are similar to starburst cells of the rabbit retina. They may participate in on and off pathways to ganglion cells and their lamination suggests that they are cholinergic.     

Localization of [F]fluorodeoxyglucose in mouse brain neurons with micro-autoradiography

This is the first study of micro-autoradiography (micro-ARG) for [¹⁸F]2-fluoro-2-deoxy- -glucose ([¹⁸F]FDG). The localization of [¹⁸F]FDG was demonstrated in dendrites of neuron and also in the myelinated axon in mouse normal brain in vivo. The nucleolus was relatively free of label. The counted silver grain numbers in autoradiogram were linearly correlated to the ¹⁸F radioactivities in the specimen. The micro-ARG using positron emitting ¹⁸F is a very time-saving technique with 4 hours exposure compared with the conventional method using ³H- or ¹⁴C-labeled tracers.     

Assessment of different methods for staining Helicobacter pylori in endoscopic gastric biopsies

The recent implication of Helicobacter pylori in the pathogenesis of gastritis-peptic ulcer syndrome and its relevance for the development of upper gastrointestinal malignancy warrant efficient methods for the detection and demonstration of the organism in biopsy specimens. We have compared 5 staining methods, namely, haematoxylin and eosin (H & E), immunohistochemistry (IHC), the silver staining HpSS, the alcian yellow-toluidine blue (Leung) method (A-Y) and Genta staining, for the demonstration of the organism in gastric biopsies taken from antrum, body and fundus of 118 patients who presented to our hospital with upper gastrointestinal symptoms. We found no significant differences in the efficacy of H & E, IHC, HpSS and A-Y in the demonstration of H. pylori in all 3 gastric sites. The least reproducible stain in our hands was the Genta stain. We conclude that H & E is adequate for the initial assessment of gastric biopsies in symptomatic upper gastrointestinal patients. This is because it is a well-tested, cheap and easy staining method, requiring a relatively short period of time to perform, with highly reproducible results. It has an added advantage of enabling simultaneous assessment of morphological changes accompanying H. pylori infection. When the density of the organism is expected to be low, we recommend addition of HpSS staining because of its high sensitivity and low cost. The disadvantages of the other staining methods (IHC, A-Y and Genta) are discussed.     

组织培养对乳胰B细胞功能和形态的影响

本文报道了不同培养时间对乳胰组织培养后胰岛活性的影响。通过对各组培养液胰岛素含量的测定和使用免疫组化与显微图象分析技术对 B 细胞内胰岛素的定性和定量观察表明,新鲜乳胰碎片行短期组织培养(2～8天),可获得良好的效果。