Control of cerebellar granule cell output by sensory-evoked Golgi cell inhibition

Classical feed-forward inhibition involves an excitation–inhibition sequence that enhances the temporal precision of neuronal responses by narrowing the window for synaptic integration. In the input layer of the cerebellum, feed-forward inhibition is thought to preserve the temporal fidelity of granule cell spikes during mossy fiber stimulation. Although this classical feed-forward inhibitory circuit has been demonstrated in vitro, the extent to which inhibition shapes granule cell sensory responses in vivo remains unresolved. Here we combined whole-cell patch-clamp recordings in vivo and dynamic clamp recordings in vitro to directly assess the impact of Golgi cell inhibition on sensory information transmission in the granule cell layer of the cerebellum. We show that the majority of granule cells in Crus II of the cerebrocerebellum receive sensory-evoked phasic and spillover inhibition prior to mossy fiber excitation. This preceding inhibition reduces granule cell excitability and sensory-evoked spike precision, but enhances sensory response reproducibility across the granule cell population. Our findings suggest that neighboring granule cells and Golgi cells can receive segregated and functionally distinct mossy fiber inputs, enabling Golgi cells to regulate the size and reproducibility of sensory responses.Classical feed-forward inhibition (FFI) involves a sequence of excitation rapidly terminated by inhibition. This temporal sequence narrows the time window for synaptic integration and enforces precise spike timing (1–7). FFI is thought to be important for regulating the temporal fidelity of spike responses in many neural systems, including the motor system, where rapid and adaptable changes in muscle activity are essential for coordinated motor control (8–10). The cerebellum plays a central role in fine sculpting of movements, and damage to the cerebellum produces severe motor deficits, most notably enhanced temporal variability of voluntary movements (11, 12). These findings suggest that cerebellar circuits have the ability to preserve precise timing information during behavior (5, 6, 13), and in vitro studies have shown that feed-forward inhibitory networks in the input layer of the cerebellum provide a mechanism for maintaining the temporal fidelity of information transmission (6, 14, 15).Synaptic inhibition in the granule cell layer is generated by Golgi cells, GABAergic interneurons that provide direct inhibitory input to granule cells (6, 15–17). The prevailing view is that, when mossy fibers are activated, granule cells receive both monosynaptic excitation and disynaptic FFI from Golgi cells, providing temporally precise inhibitory input that narrows the window for the temporal summation of discrete mossy fiber inputs (6, 14, 18). This classical excitation–inhibition sequence forms the basis of a variety of contemporary cerebellar models (7, 9, 18, 19). However, the exact temporal relationship between sensory-evoked excitation and inhibition in granule cells has never been determined in vivo. Here, we combined in vivo whole-cell voltage-clamp recordings from granule cells and in vitro dynamic clamp experiments to investigate both the temporal dynamics of Golgi-cell–mediated inhibition and its importance for shaping sensory responses in the input layer of the cerebellum.     

CD5 positive immunoregulatory B cell subsets

B-chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease often expressed as a clonal expansion of CD5+ B cells. We report the characterization of CD5+ B cells from two unique B-CLL patients. Cells from patient 1 coexpressed CD5 (leu-1), CD19 (Leu-12), CD20 (B1), and HLA-DR; they were CD10 (J5), CD21 (B2), CD22 (Leu-14), CD25 (IL2-R1), PCA-1, surface, and cytoplasmic Ig negative. They suppressed normal peripheral blood lymphocyte (PBL) pokeweed mitogen (PWM) -stimulated immunoglobulin (Ig) synthesis greater than 80%. Cells from patient 2 were CD5 (Leu-1), CD19 (Leu-12), CD20 (B1), CD21 (B2), CD22 (Leu-14), HLA-DR, IgM, and kappa positive. They were negative for CD10 (J5), CD25 (IL2-R1), and PCA-1. These cells did not suppress normal PBL PWM-stimulated Ig synthesis but produced a monoclonal IgM kappa protein with rheumatoid factor-like activity. These observations suggest that there are different CD5+ B cell subsets, one immunosuppressive and the other autoreactive.     

壁层上皮细胞的研究

近年来,随着对壁层上皮细胞(parietal epithelial cells,PECs)认识的加深,发现PECs发挥着重吸收蛋白、滤过屏障等重要功能,而且在肾脏受到损伤时,PECs可能作为潜在的干细胞,促进肾脏修复。本文就PECs及相关进展作一综述。     

Present and future cell therapies for pancreatic beta cell replenishment

If only at a small scale, islet transplantation has successfully addressed what ought to be the primary endpoint of any cell therapy: the functional replenishment of damaged tissue in patients. After years of less-than-optimal approaches to immunosuppression, recent advances consistently yield long-term graft survival rates comparable to those of whole pancreas transplantation. Limited organ availability is the main hurdle that stands in the way of the widespread clinical utilization of this pioneering intervention. Progress in stem cell research over the past decade, coupled with our decades-long experience with islet transplantation, is shaping the future of cell therapies for the treatment of diabetes. Here we review the most promising avenues of research aimed at generating an inexhaustible supply of insulin-producing cells for islet regeneration, including the differentiation of pluripotent and multipotent stem cells of embryonic and adult origin along the beta cell lineage and the direct reprogramming of non-endocrine tissues into insulin-producing cells.     

肺结核患者外周血记忆性T细胞及B细胞亚群的检测分析

目的探讨肺结核患者外周血记忆性T及B细胞亚群的表达水平。方法采用流式细胞仪检测肺结核患者（TB）、潜伏感染者（LTBI）、健康人（HD）外周血记忆性T、B细胞亚群水平及B细胞表达水平;利用Elispot技术检测外周血单个核细胞（PBMC）结核菌抗原特异性IFN-r分泌水平。结果流式结果显示,TB及LTB1的CD4＊及CD8＊中心记忆性T细胞（Tcm）显著增多,以CD4＊尤为显著;相反,效应记忆性T细胞（Tem）减少。TB记忆性B细胞均低于HD和LTBI（P〈0．01）;而在HD与LTBI及TB这三组CD19＆细胞占淋巴细胞比例中都有明显差异（P〈0．01;P〈0．001）。CD4’／CD8’Tcm与IFN-γ ELISPOT结果成正相关。结论肺结核患者外周血中存在着记忆性T及B细胞亚群的紊乱,中心记忆性T细胞增多,效应记忆性T细胞减少,记忆性T、B细胞的数量及免疫状态与结核菌感染的不同转归有关,记忆性T、B细胞在结核病发病机制中发挥着重要作用。     

Stem cell therapy in cardiovascular disorders

Heart insufficiency remains the leading cause of death despite pharmacological and interventional therapy as well as primary and secondary prevention. Laboratory research on cardiac repair implementing stem cells and progenitor cells has raised great expectations as well as controversies. The potential of diverse progenitor cells to repair damaged heart tissue includes replacement (tissue transplant), restoration (activation of resident cardiac progenitor cells, paracrine effects), and regeneration (stem cell engraftment forming new myocytes). Based on promising experimental results clinical trials including several hundreds of patients with ischemic heart disease have been initiated using mostly bone marrow-derived cells. Probably, due to a lack of standardization of cell isolation and delivery methods these trials showed controverse results regarding effectiveness. However, significant therapeutic regeneration of human myocardium could not be proven until now. Several issues are at debate concerning the translation of the experimental data into the clinic discussing the adequate cell type, dosing, timing, and delivery mode of myocardial stem cell therapy. This review focuses on the potential and clinical translation of cell based therapies in cardiovascular disease.     

Notch signaling in hematopoietic cell transplantation and T cell alloimmunity

Notch signaling can regulate both hematopoietic progenitors and alloimmune T cells in the setting of allogeneic bone marrow or hematopoietic cell transplantation (allo-HCT). Ex vivo culture of multipotent blood progenitors with immobilized Delta-like ligands induces supraphysiological Notch signals and can markedly enhance progenitor expansion. Infusion of Notch-expanded progenitors shortened myelosuppression in preclinical and early clinical studies, while accelerating T cell reconstitution in preclinical models. Notch also plays an essential role in vivo to regulate pathogenic alloimmune T cells that mediate graft-versus-host disease (GVHD), the most severe complication of allo-HCT. In mouse allo-HCT models, Notch inhibition in donor-derived T cells or transient blockade of Delta-like ligands after transplantation profoundly decreased GVHD incidence and severity, without causing global immunosuppression. These findings identify Notch in T cells as an attractive therapeutic target to control GVHD. In this review, we discuss these contrasting functions of Notch signaling with high translational significance in allo-HCT patients.     

Perfused Kupffer cell mass

The perfused Kupffer cell mass determines sulfur colloid distribution by liver spleen scan (LSS) and is proportional to the perfused hepatocyte mass. This accounts for the correlation of sulfur colloid distribution with tests of hepatic function and raises the question of whether the LSS can be used as a quantitative test of hepatic function. The recent ability to precisely measure sulfur colloid distribution by single-photon-emission computerized tomography (SPECT) prompted us to evaluate the clinical value in 329 consecutive patients with adequate LSS and clinical information, of which 27 apparent normals and 220 patients with chronic liver disease (CLD) were included in this study. The liver-bone marrow index (LBI) indicated the distribution of counts between the liver and bone marrow. The liver-spleen index (LSI) indicated the distribution between liver and spleen adjusted for spleen size. The LBI and LSI correlated with each other (r=0.753;P<0.001). The arithmetic mean of LBI and LSI was defined as the severity score. Detailed clinical evaluation was available in these patients and included 109 who had liver biopsy. A severity score in 27 normals was 102±5 (mean ±sd) with all values >85. The severity score correlated with hepatic fibrosis (r=–0.694;P<0.001) in 109 patients with benign liver disease who had recent biopsies and with the Child-Pugh classification (r=0.78;P<0.001) in 220 patients with CLD. Furthermore, 68/70 (97%) of patients with ascites, variceal bleeding, or death from hepatic failure had a severity score <85, and all patients with severity score <60 had one of these complications. All patients with liver failure had a severity score <60 and 67.7% with a score <40 died of hepatic failure. By contrast, 99.4% of patients with minimal liver disease had a severity score >80 and 96.5% a severity score >85. Mortality, the frequency of complications of CLD, and the degree of hepatic fibrosis were inversely related to the severity score. Thus, the data support the value of the LSS in detecting the perfused Kupffer cell mass and the potential for use of the LSS as a quantitative test of hepatic function.     

Multiple myeloma: reduced plasma cell contamination in peripheral blood progenitor cell collections performed after repeated high-dose chemotherapy courses

The possibility of reducing tumour cell contamination by cytotoxic drug courses prior to peripheral blood progenitor cell (PBPC) collection was evaluated in two consecutives groups of multiple myeloma (MM) patient candidates for autograft. All patients were at disease onset and received two VAD (vincristine, doxorubicin and dexamethasone) courses as initial debulking. In the first group (44 patients), mobilization and harvest were performed ‘upfront’, after a single cyclophosphamide (CY) administration of 4 g/m²; in the second group (17 patients), PBPC were collected at the end of a high-dose sequential chemotherapy programme, including: CY 5 g/m², etoposide (VP16) 2 g/m², a chemotherapy-free interval with three courses of high-dose dexamethasone, a final mobilizing CY at 7 g/m². G-CSF was given following each high-dose cytotoxic drug. Cytofluorimetric analysis was performed to quantify progenitors (CD34⁺ cells) and plasma cells, identified by the high CD38 expression and/or CD38 and CD138 coexpression. Large amounts of PBPC were collected in either group (median harvested CD34⁺/kg: 15.8 × 10⁶ and 13.4 × 10⁶, respectively; P = 0.9). Circulating plasma cells were significantly higher in patients mobilized ‘upfront’ compared to those who received the high-dose sequence (median peak values of CD38^bright/μl: 39 and 10, respectively; P = 0.02); a similar difference was observed in the amount of con-taminating plasma cells in the harvest products (median CD38^bright/kg: 7.4 × 10⁶ and 1.3 × 10⁶, respectively; P = 0.02). The results demonstrate that an in vivo purging approach is feasible in myeloma patients through repeated high-dose chemotherapy courses; this may provide less-contaminated material suitable for further in vitro purging procedures.     

Stem and progenitor cell systems in liver development and regeneration

The liver comprises two stem/progenitor cell systems: fetal and adult liver stem/progenitor cells. Fetal hepatic progenitor cells, derived from foregut endoderm, differentiate into mature hepatocytes and cholangiocytes during liver development. Adult hepatic progenitor cells contribute to regeneration after severe and chronic liver injuries. However, the characteristics of these somatic hepatic stem/progenitor cells remain unknown. Culture systems that can be used to analyze these cells were recently established and hepatic stem/progenitor cell‐specific surface markers including delta‐like 1 homolog (DLK), cluster of differentiation (CD) 13, CD133, and LIV2 were identified. Cells purified using antibodies against these markers proliferate for an extended period and differentiate into mature cells both in vitro and in vivo. Methods to force the differentiation of human embryonic stem and induced pluripotent stem (iPS) cells into hepatic progenitor cells have been recently established. We demonstrated that the CD13⁺CD133⁺ fraction of human iPS‐derived cells contained numerous hepatic progenitor‐like cells. These analyses of hepatic stem/progenitor cells derived from somatic tissues and pluripotent stem cells will contribute to the development of new therapies for severe liver diseases.     

Participation of CD45, NKR-P1A and ANK61 antigen in rat hepatic NK cell (pit cell)mediated target cell cytotoxicity

AIM Several triggering receptors have been described to be involved in natural killer (NK) cellmediated target cytotoxicity. In these studies, NK cells derived from blood or spleen were used. Pit cells are liver-specific NK cells that possess a higher level of natural cytotoxicity and a different morphology when compared to blood NK cells. The aim of this study was to characterize the role of the NK-triggering molecules NKR-P1A, ANK61 antigen, and CD45 in pit cell-mediated killing of target cells. METHODS 51 Cr-release and DNA fragmentation were used to quantify target cell lysis and apoptosis, respectively. RESULTS Flow cytometric analysis showed that pit cells expressed CD45, NKR-P1A, and ANK61 antigen. Treatment of pit cells with monoclonal antibody ( mAb ) to CD45 ( ANK74 ) not only inhibited CC531s or YAC-1 target lysis but also apoptosis induced by pit cells. The mAbs to NKRP1A (3.2.3) and ANK61 antigen (ANK61) had no effect on pit cell-mediated CC531s or YAC-1 target cytolysis or apoptosis, while they did increase the Fcγ receptor positive (FcγR+) P815 cytolysis and apoptosis. This enhanced cytotoxicity could he inhibited by 3,4-dichloroisocoumarin, an inhibitor of granzymes. CONCLUSION These results indicate that CD45 participates in pit cell-mediated CC531s and YAC-1 target cytolysis and apoptosis. NKR-P1A and ANK61 antigen on pit cells function as activation structures against FcγR+ P815 cells, which was mediated by the perforin/granzyme pathway.     

老年人巨细胞动脉炎的炎性细胞浸润类型及其分布

目的　研究老年人巨细胞动脉炎的炎性细胞浸润类型及其分布特征。方法　用免疫组织化学技术检测 2 0例巨细胞动脉炎患者颞动脉活检标本T淋巴细胞、B淋巴细胞、巨噬细胞、多核巨细胞和粒细胞表达的情况。以常规HE、美蓝染色及铅铀双重染色 ,在光镜和电镜下观察病理变化 ,比较病理变化与炎性细胞类型及分布的关系。结果　2 0例颞动脉活检标本中 ,浸润的炎性细胞由T淋巴细胞、巨噬细胞和粒细胞构成 ,构成比分别为 40 .5 %、33.5 %和 2 6 .0 %,未见B淋巴细胞和多核巨细胞。炎性细胞分布于颞动脉壁及其周围组织 ,以外膜最明显、内膜次之、中膜居末 ;有明显跳跃现象。炎性细胞分布与病理变化为非比例关系 ,病理损害以内膜明显。结论　老年人巨细胞动脉炎其颞动脉活检标本中可能有较多的粒细胞浸润 ,无多核巨细胞。     

内皮祖细胞与低氧性肺动脉高压

低氧性肺动脉高压(HPH)是一种致死性很高的肺血管疾病,其发病与血管内皮的损伤密切相关.近年来研究表明,内皮祖细胞(EPCs)在HPH血管内皮修复中具有重要作用.本文总结了近年来EPCs动员、归巢的分子生物学研究进展.研究了EPCs在HPH发生、发展中的作用,以及药物和EPCs移植对HPH的影响.     

Crosstalk between intestinal epithelial cell and adaptive immune cell in intestinal mucosal immunity

Constantly challenged by luminal bacteria, intestinal epithelium forms both a physical and biochemical defense against pathogens. Besides, intestinal epithelium senses dynamic and continuous changes in luminal environment and transmits signals to subjacent immune cells accordingly. It has been long accepted that adaptive immune cells fulfill their roles partly by modulating function of intestinal epithelial cells. Recent studies have brought up the proposal that intestinal epithelial cells also actively participate in the regulation of adaptive immunity, especially CD4+ adaptive T cells, which indicates that there is reciprocal crosstalk between intestinal epithelial cells and adaptive immune cells, and the crosstalk may play important role in intestinal mucosal immunity. This Review makes a comprehensive summary about crosstalk between intestinal epithelial cells and CD4+ adaptive T cells in intestinal immunity. Special attention would be given to their implications in inflammatory bowel disease pathogenesis and potential therapeutic targets.     

Alterations in islet cell ultrastructure following streptozotocin-induced diabetes in the rat

Summary The response of islet cells to streptozotocin-induced diabetes was investigated by transmission electron microscopy. Rats were given streptozotocin i.p. (100 mg/kg followed by another dose of 25 mg/kg 24 h later) and sacrificed after 10 days. Pancreata from rats with high serum glucose values (381–553 mg/100 ml) were selected for ultrastructural examination. Islets in these animals were smaller and much less frequent than those of control animals. Cell types were not always easily identifiable in diabetic animals: distinction between A and B cells was often difficult. Cells of some islets displayed diminished electron density and less structured cytoplasm, yet nuclei were not pyknotic. Such cells were usually deficient in secretion granules. A cells were least affected. Cells in the centers of some islets possessed multivesicular bodies, prominent dilated Golgi cisternae and secretory granules which were often pleomorphic. Nuclear envelopes of some peripheral islet cells were swollen and distended; mitochondria were enlarged and the rough endoplasmic reticulum prominent. Some islet cells retained features of B cells in spite of marked ultrastructural alterations. This diversity of morphological findings contrasts with the homogeneity of the population of streptozotocin-diabetic rats with respect to metabolic responses. Supported by Research Service Award 5T32 CA 09125 from the National Cancer Institute, DHEW.     

重组人生长激素对大鼠骨髓间质细胞作用的实验研究

目的 探讨重组人生长激素(rhGH)在体外对骨髓间质细胞(MSCs)的作用及机制.方法 四甲基偶氮唑盐微量酶反应比色法测定不同浓度、不同时间点rhGH对MSCs作用的光密度值;逆转录-聚合酶链反应测定rhGH作用及去除rhGH作用后胰岛素样生长因子1(IGF-1)mRNA的表达.结果 rhGH在10 μg/L浓度以上对MSCs有促生长作用,最佳浓度为200 μg/L,生长率为144.74%;200 μg/L的rhGH在24 h始促MSCs生长,随时间延长逐渐增强,72 h生长率为144.10%.IGF-1mRNA含量在rhGH作用24、48和72 h逐渐增加,去除rhGH后IGF-1 mRNA表达虽有所减少,但仍随时间延长逐渐增加,2周达高峰.结论 rhGH在体外可促进MSCs生长,其最适浓度为200 μg/L;IGF-1介导是rhGH促MSCs生长的途径之一.