Structure and Histochemistry of the Skin of a Torrent Catfish, Liobagrus mediadiposalis

The epidermis of the torrent catfish, Liobagrus mediadiposalis, consists of three layers: the outermost layer, middle layer and stratum germinativum. The epidermis consists of two types of skin glands, small mucus cell and voluminous club cell. The unicellular mucus cell contains acid sulfomucins (some sialomucins) and the club cell, sometimes binucleate, is proteinaceous. Well-developed vascularization is one of the characteristics of epidermis of L. mediadiposalis. Well-developed lymphatic spaces contain lymphocytes in the epidermis. The dermis lacks scales and consists mostly of a thick, dense connective tissue; its superficial region just below the basal membrane is supplied with fine blood capillaries. These histological features of the skin in L. mediadiposalis are consistent with that required for cutaneous respiration.     

C-Med 100®, a hot water extract of Uncaria tomentosa, prolongs lymphocyte survival in vivo

Water extracts of the bark of Uncaria tomentosa, a vine indigenous to South America, has been used for generations as an "immuno modulator". To understand the basis of this immuno modulatory effect we fed mice in their drinking water with C-Med 100, which is a commercially available water extract from Uncaria tomentosa. We found a dose-dependent increase in spleen cell numbers in the supplemented mice, but the proportions of B cells, T cells, NK cells, granulocytes, and memory lymphocytes were normal. However, there were no detectable changes of the lymphoid architecture of the spleen even after long-term treatment. Further, when C-Med 100 treatment was interrupted the cellularity returned to normal level within four weeks. The increased number of lymphocytes was most likely not due to increased production because C-Med 100 did not have any significant effect on precursor cells nor on the accumulation of recent thymic emigrants in the spleen. We conclude that accumulation is most likely due to prolonged cell survival, because adoptive transfer experiments demonstrated that C-Med 100 treatment significantly prolonged lymphocyte survival in peripheral lymphoid organs, without increasing their proliferation rate. Since the accumulation was reversible and without detectable pathological effects, these results suggest the use of C-Med 100 as a potential agent for clinically accelerating the recovery of patients from leukopenia.     

Granzymes, cytotoxic granules and cell death: the early work of Dr. Jurg Tschopp

Within the powerful legacy left by Jurg Tschopp, we should not forget his early work that helped to elucidate the molecular pathways responsible for the clearance of virus-infected and transformed cells by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Jurg's skilful biochemical approach formed a firm platform upon which the work of so many other biochemists, cell biologists and immunologists would come to rely. Jurg coined the shorthand term 'granzyme' to denote the individual members of a family of serine proteases sequestered in and secreted from the cytotoxic granules of CTL/NK cells. He was also one of the first to describe the lytic properties of purified perforin and to postulate the synergy of perforin and granzymes, which we now know to underpin target cell apoptosis. Jurg was a major protagonist in the debate that raged throughout the 1980's and early 1990's on the physiological relevance of the 'granule exocytosis' pathway. Ultimately, resolving this issue led Jurg and his colleagues to even greater and impactful discoveries in the broader field of apoptosis research. Jurg Tschopp ranks with other pioneers, particularly Gideon Berke, Chris Bleackley, Pierre Golstein, Pierre Henkart and Eckhard Podack for making seminal discoveries on our understanding of how the immune system eliminates dangerous cells.     

甲状腺激素对适应性免疫细胞的作用

摘要：甲状腺激素（Thyroid hormones,THs）参与免疫功能的调节,在固有免疫和适应性免疫中发挥着重要作用。THs异常分泌所致的免疫功能失调被认为参与了格雷夫斯病和桥本甲状腺炎等自身免疫性疾病的发生发展。目前,THs在固有免疫细胞（中性粒细胞、巨噬细胞、树突状细胞、自然杀伤细胞、肥大细胞）中的作用已得到了较好的阐明,但THs对适应性免疫细胞（T淋巴细胞与B淋巴细胞）的影响等方面的研究仍未引起足够的重视。因此,本研究从适应性免疫细胞的角度出发,重点讨论了THs对这些细胞的发育、分化及功能等方面的影响,为进一步理解THs调节免疫功能的作用提供新视角。     

EB病毒诱导的胸腺恶性T细胞淋巴瘤细胞体外长期培养研究

建立含有EB病毒的T细胞淋巴瘤细胞系,为探讨EB病毒的致瘤机理,研究EB病毒在T细胞淋巴瘤发生过程中的作用提供手段.在TPA协同EB病毒诱导胸腺恶性T细胞淋巴瘤动物模型的基础上,联合应用IL-2,将诱导的肿瘤组织进行体外细胞培养,成功地分离获得一株在体外长期存活的淋巴细胞TET.T细胞亚群分类实验证实TET细胞为CD4阳性的T淋巴细胞,PCR和原位杂交可检测到EB病毒的EBERs、LMP1和BARF1,并有LMP1蛋白的表达.TET细胞的获得,有望在体外建立转化细胞系,为体外研究EB病毒的致瘤机理及防治提供理想的实验材料.     

Immunization with dendritic cells loaded with alpha-galactosylceramide at priming phase, but not at boosting phase, enhances cytotoxic T lymphocyte activity against infection by intracellular bacteria

We evaluated the effect of immunization with dendritic cells (DCs) pulsed with alpha-galactosylceramide (alphaGalCer) and listeriolysin O (LLO) 91-99 peptide, a dominant cytotoxic T lymphocyte (CTL) epitope of Listeria monocytogenes by observing the responses of specific CD8(+) T cells and in vivo CTL activity. DCs were pulsed with various combinations of alphaGalCer and LLO91-99 peptide and administered to BALB/c mice. Immunization with DCs pulsed with alphaGalCer and LLO91-99 at priming phase and with DCs pulsed with LLO91-99 alone at boosting phase induced stronger in vivo CTL activity, reduced the bacterial load in spleens of Listeria-challenged mice and augmented CD62L(+) CD8(+) central memory T cells compared with other immunization protocols. The blockade of interferon-gamma (IFN-gamma) at boosting phase reversed the induction of CD8(+) central memory T cells and reduced the bacterial load in spleens of Listeria-challenged mice immunized with DCs pulsed with alphaGalCer and LLO91-99 at both phases, suggesting that alphaGalCer at boosting phase has deleterious effects through IFN-gamma production. These results indicate that immunization with DCs pulsed with CTL epitope peptide together with alphaGalCer at priming phase, but not at boosting phase, is feasible for eliciting a specific CTL activity and protective immunity against infection of intracellular bacteria.     

蛇胸腺胚胎发育的组织学研究

该文应用光镜、电镜和细胞计数技术对胚胎发育期虎斑颈槽蛇胸腺的发育分化进行了研究。在胚胎发育１１期,胸腺原基内出现前淋巴细胞。从胚胎发育１２期至出生前（１６期）,淋巴细胞不断增殖分化,小淋巴细胞逐渐增多,而淋巴母细胞和中淋巴细胞逐渐减少。胸腺皮质和髓质形成于１６期。巨噬细胞以及肌样细胞和胸腺ＡＰＵＤ细胞分别形成于胚胎发育１４期和１５期,随后数量有所增加,分别分布于胸腺皮质和髓质。     

Expression and purification of a soluble B lymphocyte stimulator mutant modified with the T-helper cell epitope

The DNA encoding soluble B lymphocyte stimulator (134–285 amino acids, sBLyS) mutant with residues 217–224 replaced by two glycines (named msBLyS) was constructed. The sequence encoding a foreign immunodominant T-helper epitope from ovalbumin (OVA) was then coupled to the 5′-end of msBLyS cDNA. After being sequenced, the recombinant DNA was ligated into the prokaryotic expression vector pQE-80L. The recombinant protein was produced in E. coli DH5α after induction with IPTG with the yield of more than 40% of total bacterial protein. The recombinant protein was purified with Ni-NTA chromatography and Sepharcryl S200 chromatography to a purity of more than 98%. The BALB/c mice, immunized with the recombinant protein, produced anti-BLyS antibodies at a high level, which indicated that the recombinant BLyS mutant modified with T-helper epitope elicited polyclonal antibodies with cross-reactivity with BLyS in vivo. This recombinant protein may therefore be used as immune inhibitor of BLyS for treating BLyS -associated autoimmune diseases.     

Highly efficient gene transfer into antigen-specific primary mouse lymphocytes with replication-deficient retrovirus expressing the 10A1 envelope protein

Background

Introduction of recombinant genes in the genome of primary lymphocytes by virtue of a replication‐deficient retrovirus can be used in immunological studies and for cell‐based gene therapy.

Methods

Packaging cells GP+E86 producing replication‐deficient retrovirus incorporating the genes of enhanced green fluorescent protein (eGFP), C2γ or C2ξ, were generated by calcium phosphate‐mediated transfection. Clones with the highest titres of retrovirus vector were isolated from them and their supernatants were used for transduction of PT67 cells. Primary mouse lymphocytes and T‐cell hybridoma MD.45 were transduced by centrifugation with retroviral stock. The retroviral content of packaging cell supernatants was determined by dot blotting and hybridization with a DNA probe.

Results

PT67 cells produced ～50 times more retrovirus vector than the original GP+E86 clones. When retroviral stocks of PT67 and GP+E86 cells were used at 1/50 dilution and undiluted, respectively (to normalize them forretroviral RNA content), the transduction efficiency of mouse T‐cell hybridoma was 40% and 5%, respectively. Centrifugation of target cells with retroviral stock at 2000 g for 60 min increased the percentage of transduced cells two‐ to three‐fold. Within a population of cells isolated from the draining lymph nodes of an immunized mouse and reactivated with an antigen, up to 60% of CD4⁺ T cells and up to 80% of B cells could be transduced with a transgene in replication‐deficient retrovirus packaged by PT67 cells using the optimized gene transfer protocol.

Conclusions

This protocol allows for the generation of packaging cells producing high titres of retrovirus vector. The 10A1 envelope protein is superior to the ecotropic one for the transduction of mouse lymphocytes. Copyright © 2002 John Wiley & Sons, Ltd.

     

Pre-B cell colony enhancing factor (PBEF), a cytokine with multiple physiological functions

Pre-B cell colony enhancing factor (PBEF) is regarded as a proinflammatory cytokine. Named for its first discovered function as a pre-B cell colony enhancing factor, it has since been found to have many other functions relating to cell metabolism, inflammation, and immune modulation. It has also been found to have intracellular and extracellular forms, with the two overlapping in function. Most of the intracellular functions of PBEF are due to its role as a nicotinamide phosphoribosyltransferase (Nampt). It has been found in human endothelial cells, where it is able to induce angiogenesis through upregulation of VEGF and VEGFR and secretion of MCP-1. In human umbilical endothelial cells, PBEF increases levels of the protease MMP 2/9. PBEF has also been found in a variety of immune cells other than B cells and has been shown to inhibit apoptosis of macrophages. Extracellular PBEF has been shown to increase inflammatory cytokines, such as TNF-α, IL-1β, IL-16, and TGF-β1, and the chemokine receptor CCR3. PBEF also increases the production of IL-6, TNF-α, and IL-1β in CD14⁺ monocyctes, macrophages, and dendritic cells, enhances the effectiveness of T cells, and is vital to the development of both B and T lymphocytes. The purpose of this review is to summarize the recent advances in PBEF research.     

Cytotoxic activity and T cell receptor repertoire in tumor-infiltrating lymphocytes of adrenal cell carcinomas

The cytotoxic activity and T cell receptor (TCR) V repertoire in tumor-infiltrating lymphocytes (TIL) of three primary adrenal cell carcinomas were analyzed. Fresh, non-cultured TIL from two of the three tumors showed low but significant lysis of the autologous tumor, and for one of the patients this activity was strongly enhanced upon culture in interleukin-2. An allogeneic adrenal cell carcinoma line and the K562 or Daudi targets included as controls were not killed. Phenotypic analysis of freshly isolated TIL demonstrated that the cells from the two patients that demonstrated cytolytic capacity mainly consisted of CD45RO⁺ T cells. In vitro cultured TIL lines from these patients demonstrated a high percentage of CD8⁺ cells expressing either the V6 gene or the V8 gene product, as measured with a panel of mAb specific for TCR V and V gene products. Analysis of the TCR V gene mRNA expression in freshly isolated non-cultured TIL, using a polymerase-chain-reaction-assisted cDNA-amplification assay, confirmed the strong expression of the genes coding for the TCR V6 or the V8. This assay also demonstrated a more restricted TCR V gene usage in the TIL as compared to peripheral blood lymphocytes from the same patient.This study was supported by the Swedish Cancer Society and by the Cancer Society in Stockholm     

Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European ‘Biorack’ provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the ‘Biorack’ facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatent in-flight), injection port, and supernatent collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatent, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground- based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities. J. Cell. Biochem. 70:252–267, 1998. © 1998 Wiley-Liss, Inc.     

人外周血活化淋巴细胞survivin基因的转录激活和表达相关的信号转导通路研究

Survivin是与细胞增殖和细胞凋亡调控密切相关的重要功能蛋白质,也是肿瘤临床诊断的分子标志物及治疗研究的理想靶标.由于发现人外周血活化淋巴细胞存在Survivin蛋白的显著表达,故以植物血凝素(PHA)和IL-2共刺激培养的正常人外周血单个核细胞为实验材料,采用RT-PCR、蛋白质印迹以及细胞周期分析等实验方法,观察淋巴细胞活化与Survivin蛋白表达之间的相互关系,并利用3种激酶抑制剂探索了淋巴细胞活化所致Survivin蛋白表达相关的信号转导通路.实验结果表明：人外周血单个核细胞在刺激培养36 h前后出现survivin基因的明显转录激活和蛋白质的起始表达,表达产物的水平随培养时间的延长而增加,且具有明显的细胞周期和周期时相依赖性.JAK2的抑制剂AG490阻断细胞周期的运行,且强烈抑制survivin基因的转录激活和蛋白质表达,PI3K和MEK的抑制剂Wortmannin和PD98059也有一定程度的抑制作用.所得实验结论是：survivin基因的转录激活和蛋白质表达与淋巴细胞活化相关联,代表细胞处在增殖状态.JAK2介导的JAK-STAT信号通路是淋巴细胞活化,Survivin蛋白表达必需和最重要的信号转导通路,PI3K和MEK介导的信号通路也具有一定的影响作用.     

Suppression of lymphocyte proliferation by a nonspecific factor produced by the burkitt lymphoma-derived lymphoblast cell line

Summary The DAUDI lymphoblast cell line derived from a patient with Burkitt lymphoma was obtained from two different sources. One of these (DAUDI-I) produced a factor that inhibited lymphocyte proliferation in both human and mouse regardless of the stimulator, i.e. allogeneic lymphocytes or mitogens. Glutaraldehyde treatment eliminated production of the factor and demonstrated that DAUDI-I was capable of stimulating normal lymphocytes in MLR. A second DAUDI cell line (DAUDI-S) did not produce the inhibitory factor and was capable of MLR stimulation. Supported by the Children's Leukemia Foundation of Michigan, NIH Grants AI 11013 and AI 11335, and the Kidney Foundation of Michigan.     

磁性细胞分选技术的应用与生物学评价

磁性细胞分选技术是一种利用超顺磁性纳米复合材料进行细胞分选的细胞高度特异性快速分选技术,在免疫学、干细胞学、肿瘤学和临床医学等领域应用广泛。本文综合阐述了磁性细胞分选技术的分类和应用,讨论了近几年出现的几项基于磁性细胞分选的新技术和面临的挑战。重点分析了磁性细胞分选产品生物学评价的必要性,并提出了10项与磁性细胞分选产品相关的生物学评价技术参数：得率、纯度、无菌、细胞毒性、细胞形态、活率、细胞的光散射特性、细胞的荧光抗体标记能力、细胞活化、细胞增殖,该评价技术参数的提出对磁性细胞分选规范化应用具有重要的推动作用。     

Gamma irradiation of human dendritic cells influences proliferation and cytokine profile of T cells in autologous mixed lymphocyte reaction

Dendritic cells (DC) are the most potent antigen-presenting cells (APC); their ability to induce proliferation of T cells in a mixed lymphocyte reaction (MLR) assay is commonly used for the evaluation of their function. It is a general thought that gamma irradiation of APC does not influence their ability to activate T-cell proliferation, but the data from several studies are controversial. To further determine the mechanisms involved in DC-induced T-cell activation in MLR assay, human DC induced from peripheral blood mononuclear cells (PBMC) were gamma-irradiated and determine their effects on the proliferation and cytokine profiles of T cells in an autologous MLR. DC were induced from the PBMC of 11 multiple sclerosis (MS) patients with RMPI 640 medium containing recombinant human GM-CSF (rhGM-CSF; 800 U/ml) and recombinant human IL-4 (rhIL-4; 500 U/ml). DC harvested on day 7 were divided into two equal parts. One part was not irradiated (naive DC); the other was gamma-irradiated at a dose of 30 Gy. Cell surface molecules were analyzed by flow cytometry. T-cell proliferation was determined using a beta-scintillation counter. The levels of IL-2, IL-4, IL-6 and IL-10 in co-culture supernatants were measured by ELISA. The results indicated that gamma irradiation reduced expression of CD86, CD80 and HLA-DR molecules on DC, especially CD86 (P=0.0072). DC, irradiated or non-irradiated, effectively stimulated autologous T-cell proliferation. Compared to naive DC, irradiated DC showed a markedly lower capacity to promote T-cell proliferation (P=0.0073), and strikingly up-regulated secretion of IL-4 (P=0.0145) and IL-2 (P=0.0323) by autologous T cells. No significant differences were noted in IL-6 and IL-10 production between T cells co-cultured with naive DC and irradiated DC (P>0.05). It is concluded that gamma irradiation of DC not only influences the phenotype of DC but also alters their capacity to stimulate the proliferation and the cytokine profiles of autologous T cells in a MLR.     

Expression,characterization of recombinant human soluble BAFF secreted from CHO cell

B cell-activating factor of the TNF family (BAFF) is critical for B cell maturation and survival. Here, we constructed a stable CHO cell line, in which the expression level of soluble form of BAFF (sBAFF) was raised from 0.13 μg/ml to 0.55 μg/ml. Purified recombinant sBAFF from these CHO cells not only bound to its receptors but also co-stimulated the proliferation of human peripheral blood B lymphocyte in vitro. These results provided us with a useful basis for further studies about sBAFF-related research.