NK,NKT and Invariant-NKT Cells in Tumor Draining Lymph Nodes of Patients with Breast Cancer

Background: NK (natural killer) and NKT (natural killer T) cells, as components of innate immune system, play a crucial role in tumor progression and dissemination. Objective: To investigate the percentages of NK cells, NKT cells, iNKT (invariant natural killer T) cells, total T lymphocytes as well as activated T lymphocytes, in tumor draining lymph nodes (TDLNs) of patients with breast cancer (BC) and their association with different clinic-pathological features of the patients. Methods: Axillary lymph nodes were obtained from 30 Iranian women with breast cancer. After routine pathological evaluations, mononuclear cells were separated from their lymph nodes and incubated with appropriate fluorochrome conjugated monoclonal antibodies specific for CD3, HLA-DR, CD16/56, and Vα24Jα18-TCR. Data were collected on a four-color flow cytometer and analyzed by CellQuest software. Results: The mean percentages of NK (CD3-CD16/56+), NKT (CD3+CD16/56+) and iNKT (Vα24Jα18-TCR+) cells in TDLNs mononuclear cells of BC patients were 2.04%, 2.44% and 0.1%, respectively. A significant decrease in the percentages of NK and iNKT subsets in patients with grade I was observed compared to grade III (p=0.03 and p=0.01, respectively). Moreover, NK cells were increased in patients with grade III of BC compared to grade II (p= 0.003). Conclusion: The increase in the percentage of NK and iNKT cells in TDLNs of patients with higher grade of BC might suggest a suppressive phenotype for these cells in breast cancer, which merit more functional investigation.     

RAW264.7细胞泡沫化前后差异蛋白质组分析

目的 应用凝胶内差异显示电泳技术和质谱技术研究小鼠巨噬细胞RAW264.7泡沫化前后蛋白质组的差异.方法 体外培养RAW264.7细胞,用氧化型低密度脂蛋白作用使其转变为泡沫细胞.分别提取RAW264.7细胞泡沫化前后的细胞总蛋白,用荧光染料Cy3或Cy5进行标记,与Cy2标记的内标等量混合后在同一胶中进行电泳分离,经不同光激发后扫描得到不同样品的蛋白质组图谱.采用DeCyder 6.5软件进行差异蛋白质组分析,筛选出12个差异蛋白质.经质谱鉴定和分析,其中10个蛋白质得到鉴定.结果 RAW264.7细胞泡沫化后,应激蛋白70、二硫键并构酶、细胞质肌动蛋白和一种未命名的蛋白质表达量降低,而葡萄糖调节蛋白、烯醇酶、Enol蛋白、Peroxiredoxin4、Stathmin 1和BID蛋白表达量上升.结论 本研究建立了巨噬细胞泡沫化前后蛋白质组图谱,并成功进行差异蛋白质组分析,从蛋白质组水平增加了对细胞泡沫化的机制认识,为细胞泡沫化形成动脉粥样斑块的干预研究提供新思路和新靶点.     

Mesenchymal Stem Cells Do Not Suppress Lymphoblastic Leukemic Cell Line Proliferation

Background: Several studies have demonstrated the immunosuppresive effects of mes-enchymal stem cells (MSCs) in allogeneic or mitogenic interactions. Cell-cell contact inhibition and secretion of suppressive soluble factors have been suggested in this re-gard. Objective: To investigate if adipose derived MSCs could inhibit Jurkat lym-phoblastic leukemia T cell proliferation during coculture. Methods: Adherent cells with the ability of cellular growth were isolated from normal adipose tissues. Initial charac-terization of growing cells by flow cytometry suggested their mesenchymal stem cell characteristics. Cells were maintained in culture and used during third to fifth culture passages. Jurkat or allogeneic peripheral blood mononuclear cells (PBMCs) were la-beled with carboxy fluorescein diacetate succinimidyl ester and cocultured with increas-ing doses of MSCs or MSC culture supernatant. Proliferation of PBMCs or Jurkat cells under these conditions was assessed by flow cytometry after 2 and 3 days of coculture, respectively. Results: Results showed the expression of CD105, CD166 and CD44, and the absence of CD45, CD34 and CD14 on the surface of MSC like cells. Moreover, ini-tial differentiation studies showed the potential of cell differentiation into hepatocytes. Comparison of Jurkat cell proliferation in the presence and absence of MSCs showed no significant difference, with 70% of cells displaying signs of at least one cell division. Similarly, the highest concentration of MSC culture supernatant (50% vol/vol) had no significant effect on Jurkat cell proliferation (p>0.6). The same MSC lots significantly suppressed the allogeneic PHA activated PBMCs under similar culture conditions. Conclusion: Using Jurkat cells as a model of leukemia T cells, our results indicated an uncertainty about the suppressive effect of MSCs and their inhibitory metabolites on tumor or leukemia cell proliferation. Additional systematic studies with MSCs of differ-ent sources are needed to fully characterize the immunological properties of MSCs be-fore planning clinical applications.     

大鼠骨髓基质干细胞体外培养方法的实验研究

目的研究培养骨髓基质干细胞(MSCs)的简易方法和其在体外经5-氮胞苷诱导为心肌细胞,为心肌梗死后心力衰竭的干细胞移植治疗提供理想的种子细胞。方法采用传统的密度梯度离心分离加贴壁法和略加改进的Wakitani方法。具体操作是:取SD大鼠分离出股骨和胫骨,用10%DMEM培养基冲出骨髓液,以2×105/ml密度接种于培养瓶中,培养出的骨髓基质干细胞连续传3代,使细胞纯化后,用5-氮胞苷诱导,观察细胞形态改变。结果大鼠骨髓液接种于培养瓶中后,第2天大部分细胞已经贴壁,贴壁生长的细胞以分散、克隆集落的方式增殖,细胞大多呈梭形,7～10d后克隆集落逐渐增多,细胞融合,传代培养后细胞呈分布均匀的纺锤形细胞生长。MSCs经10!mol/L5-氮胞苷诱导3周后,发现细胞形态较前更加细长,呈梭形,类似心肌细胞样形态。结论应用略加改进的Wakitani方法,能较好地进行骨髓基质干细胞的培养和扩增,且操作简便;在体外培养的骨髓基质干细胞经诱导可转化为心肌细胞,有望成为心肌梗死后心力衰竭干细胞移植治疗的细胞材料。     

载脂蛋白E基因缺失促进不成熟髓系细胞的增殖和向单核细胞的极化

目的 研究载脂蛋白E（ApoE）对小鼠骨髓来源的CD11b⁺Gr-1⁺髓系细胞增殖和分化的影响,阐述ApoE基因敲除小鼠（ApoE^－/－）致动脉粥样硬化敏感的炎症相关新机制。方法 6～8周龄的ApoE^－/－小鼠和C57/B6野生型小鼠,采用流式细胞术分析骨髓、脾脏和外周血中CD11b⁺Gr-1⁺ 髓系细胞、CD11b⁺Gr-1^-单核细胞和CD11b^-Gr-1⁺粒细胞的百分比的变化。应用定量RT-PCR和免疫荧光染色,鉴定ApoE基因和蛋白在CD11b⁺Gr-1⁺ 髓系细胞的表达。从骨髓分选CD11b⁺Gr-1⁺ 髓系细胞,体外培养24 h,流式细胞术分析ApoE基因缺失对髓系细胞周期改变的作用。结果 （1）ApoE基因缺失显著增加ApoE^－/－小鼠外周血CD11b⁺Gr-1⁺ 髓系细胞和CD11b⁺Gr-1^-单核细胞;（2）ApoE基因缺失促进ApoE^－/－小鼠脾脏和骨髓中CD11b⁺Gr-1⁺ 不成熟髓系细胞的增殖;（3）定量RT-PCR和免疫荧光染色证实ApoE在CD11b⁺Gr-1⁺髓系细胞有较高水平的表达;（4）ApoE基因缺失可以促进CD11b⁺Gr-1⁺细胞周期自G₁期进入S期。结论 ApoE基因缺失显著增加ApoE^－/－小鼠脾脏和骨髓中CD11b⁺Gr-1⁺ 不成熟髓系细胞的增殖、巨噬细胞分化和动员。ApoE基因缺失促进CD11b⁺Gr-1⁺ 髓系细胞增殖与其促进细胞周期进入S期有关。     

胃感觉过敏患者胃黏膜5-羟色胺阳性细胞的变化

背景:目前研究认为5-羟色胺(5-HT)参与了内脏感觉过敏的发生。已发现肠易激综合征患者肠道黏膜中分泌5-HT的肠嗜铬细胞(EC细胞)和肥大细胞有所改变。目的:研究胃感觉过敏患者近端胃黏膜5-HT阳性细胞的变化,探讨其在胃感觉过敏中的作用。方法:应用电子恒压器测定40例功能性消化不良(FD)患者和15名正常对照者的胃感觉阈值,根据胃感知阈值将23例行胃镜活检组织病理学检查的FD患者分为感觉过敏(FD-H)组和感觉正常(FD-N)组。以免疫组化方法检测近端胃黏膜5-HT阳性细胞,应用图像分析系统测定其积分光密度(IOD)值。结果:FD组的胃感知、不适和疼痛阈值均较正常对照组显著下降。FD-H组近端胃黏膜每高倍视野下5-HT阳性细胞数显著多于FD-N组和正常对照组(14.1±2.3对8.7±1.9和8.3±1.4,P<0.05)。扩张刺激后,FD-H组5-HT阳性细胞IOD值的降幅(反映介质释放量)较FD-N组和正常对照组显著增加(16.3%±3.4%对10.7%±2.2%和8.1%±2.3%,P<0.05)。直线相关分析显示,5-HT阳性细胞释放介质越多,胃感知阈值越低。结论:部分FD患者存在胃感觉过敏,与近端胃黏膜5-HT阳性细胞数量增多、介质释放增加有关。     

Acute Ethanol Intoxication Prevents Lipopolysaccharide-Induced Down Regulation of Protein Kinase C in Rat Kupffer Cells

Protein kinase (PK) C has been implicated in a number of cellular events, many of which are also known to be affected by ethanol (ETOH). ETOH intoxication is also known to impair immune function, thereby increasing the host's susceptibility to infection. The purpose of this study was to assess the effect of acute ETOH intoxication on PKC activity and its intracellular distribution in nonparenchymal liver cells following an E. coli lipopolysaccharide (LPS) challenge. The liver was chosen for the study because it is the primary site both for metabolism of ETOH and detoxification of gut derived bacterial products. Catheterized conscious rats were administered saline or ETOH (175 mg/100 g body weight as a bolus followed by a continuous, 7 hr infusion of 28 mg/100 body weight/hr). LPS was injected intravenously (100 micrograms/100 g body weight) 3 hr before the end of the saline or ETOH infusion. Kupffer and endothelial cells were isolated by collagenase-pronase digestion followed by centrifugal elutriation. PKC was assayed after extraction with digitonin containing buffer and partial purification on DE-52 cellulose minicolumns. LPS decreased PKC activity by 69% from control values. Although ETOH infusion alone did not affect PKC activity in Kupffer cells, it completely abrogated the LPS effect. A similar trend was observed for the endothelial cells. No significant differences were observed between groups with respect to the intracellular distribution of PKC. The down-regulation of PKC by LPS may represent a mechanism of functional adaptation of the immunocompetent cells to one of the cytokines, i.e., TNF, whose receptors are down regulated by activation of PKC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term Omeprazole Treatment Suppresses Body Weight Gain and Bone Mineralization in Young Male Rats

Background: The stomach is rich in endocrine cells, including those producing ghrelin, which is thought to play a role in the control of body growth. Omeprazole treatment is associated with hypergastrinaemia, resulting in growth of the oxyntic mucosa in general and the enterochromaffin-like (ECL) cells in particular. In the present study, we examined the effects of long-term omeprazole treatment on young male rats with respect to body growth and stomach. Methods: Male rats (24 days old) were treated with omeprazole (400 µmol/kg/day) or vehicle for 77 days. The body weight was recorded twice per week. At sacrifice, dual-energy X-ray absorptiometry (DXA) was used to assess total bone area, bone mineral content (BMC), bone mineral density (BMD) and body composition (fat and lean body mass). The lengths of the spine and the femur were recorded. The plasma concentrations of gastrin and histamine were determined by radioimmunoassays. The endocrine cells of the stomach were examined by immunocytochemistry. Results: The body weight gain was suppressed by omeprazole treatment. The bone area, BMC and BMD were reduced, while the lengths of the spine and the femur and the body composition were unchanged. Omeprazole-induced hypergastrinaemia was associated with enlargement of the oxyntic area and with hyperplasia of ECL cells but not of A-like cells and D cells. In contrast, the enterchromaffin (EC) cell density in the antrum was reduced. Conclusions: Omeprazole treatment of young male rats reduces body weight and bone mass gain. The densities of ECL cells in the oxyntic mucosa was increased and of the EC cells in the antral mucosa reduced.     

Chronic ethanol consumption decreases murine Langerhans cell numbers and delays migration of Langerhans cells as well as dermal dendritic cells

Background: Chronic alcoholics experience increased incidence and severity of infections, the mechanism of which is incompletely understood. Dendritic cells (DC) migrate from peripheral locations to lymph nodes (LN) to initiate adaptive immunity against infection. Little is known about how chronic alcohol exposure affects skin DC numbers or migration. Methods: Mice received 20% EtOH in the drinking water for up to 35 weeks. Baseline Langerhans cell (LC) and dermal DC (dDC) numbers were enumerated by immunofluorescence (IF). LC repopulation after inflammation was determined following congenic bone marrow (BM) transplant and ultraviolet (UV) irradiation. Net LC loss from epidermis was determined by IF following TNF‐α or CpG stimulation. LC and dDC migration into LN was assessed by flow cytometry following epicutaneous FITC administration. Results: Chronic EtOH consumption caused a baseline reduction in LC but not dDC numbers. The deficit was not corrected following transplantation with non‐EtOH‐exposed BM and UV irradiation, supporting the hypothesis that the defect is intrinsic to the skin environment rather than LC precursors. Net loss of LC from epidermis following inflammation was greatly reduced in EtOH‐fed mice versus controls. Ethanol consumption for at least 4 weeks led to delayed LC migration into LN, and consumption for at least 8 weeks led to delayed dDC migration into LN following epicutaneous FITC application. Conclusions: Chronic EtOH consumption causes decreased density of epidermal LC, which likely results in decreased epidermal immunosurveillance. It also results in altered migratory responsiveness and delayed LC and dDC migration into LN, which likely delays activation of adaptive immunity. Decreased LC density at baseline appears to be the result of an alteration in the skin environment rather than an intrinsic LC defect. These findings provide novel mechanisms to at least partially explain why chronic alcoholics are more susceptible to infections, especially those following skin penetration.     

Rosuvastatin Intervention Decreased the Frequencies of the TIM-3+ Population of NK Cells and NKT Cells among Patients with Chronic Hepatitis B

Background: Natural killer (NK) cells are dichotomously involved in chronic hepatitis B (CHB) infection as principal members of innate immunity. An effective treatment should enhance the antiviral potentials of NK cells and not their immunomodulatory roles. TIM-3 (T-cell immunoglobulin and mucin-containing domain) is a molecule with an essential role in controlling immune tolerance. TIM-3 demonstrated the highest expression among NK cells of patients with chronic liver disorders. Statins have been reported to attenuate the levels of TIM-3 on NK cells.Objectives: To investigate the frequencies of NK cells, NKT cells, and TIM-3+ population in patients with CHB upon rosuvastatin (RSV) intervention.Methods: Thirty confirmed patients with CHB were randomly assigned into two groups of 15 (receiving 20 mg of RSV or placebo per day) for 12 weeks. We evaluated the percentages of TIM-3+ cells by staining the peripheral blood mononuclear cells (PBMCs) with CD3, CD16, and CD56 markers using flow cytometry.Results: Our findings indicated that RSV administration could increase CD3- CD56+ NK cells (P>0.05) and CD3+ CD16+ CD56+ NKT cells (P<0.05). RSV intervention could reduce the percentages of TIM-3+ cells among NK cells (P<0.01) and NKT cells (P> 0.05) of patients with CHB compared with the placebo group.Conclusions: The increased population of NK and NKT cells and the effective reduction of TIM-3+ cells among patients with CHB delineated that rosuvastatin could be proposed as an appropriate modulator of innate immune response (regarding NK and NKT cells) in favor of enhancing their antiviral activities.     

人循环内皮祖细胞不同克隆成分的体内外血管生成功能不同

目的探讨成人外周血循环来源的内皮祖细胞不同克隆成分表型特点及体内外血管生成差异。方法密度梯度离心法获得单个核细胞,用含生长因子的内皮培养基接种于纤维连接蛋白包被的培养板中。7d后计数早期克隆并进行下面实验,另1份持续培养直到晚期克隆出现进行相同实验。流式细胞术检测细胞表面抗原,间接荧光染色法鉴定细胞表达假血友病因子。胶原凝胶细胞体外种植及裸鼠体内移植实验分别测定体外及体内血管生成功能。结果早期克隆再种植不能形成第二代克隆且无体内外血管形成功能,细胞表面主要表达CD14和CD45。晚期克隆在培养21~28d间出现,再种植可形成第二代内皮细胞克隆,并能在体外和裸鼠体内胶原凝胶中形成管腔样结构,细胞表达CD45和CD14显著减少(P<0.001)而CD146明显增加(P<0.01)。结论人外周血单个核细胞在内皮培养条件下可形成早期克隆和晚期克隆,只有晚期克隆表现出干/祖细胞和内皮细胞双重表型特征并具有体内外血管生成功能。     

HepG2细胞抗原对脐血CD34^＋造血干细胞诱导分化的树突细胞免疫功能的影响

背景：树突细胞（DC）是体内功能最强的抗原呈递细胞,可激活初始型T细胞,生成辅助性T细胞和杀伤性T细胞。DC具有特异性呈递肿瘤抗原的能力,在肿瘤免疫中发挥重要作用。目的：探讨HepG2细胞抗原对脐血CD34^＋造血干细胞诱导分化的DC免疫功能的影响。方法：分离培养脐血CD34^＋造血干细胞后,加入细胞因子组合诱导生成DC并将其分成HepG2细胞抗原负载组和对照组．以流式细胞仪测定DC生成率和免疫表型,以酶联免疫吸附测定（ELISA）检测干扰素-γ（IFN-γ）含量,以MTT法检测细胞毒性T淋巴细胞（CTL细胞）对HepG2细胞的杀伤作用。结果：DC生成率为60．2％±9．4％。与对照组相比,HepG2细胞抗原负载组DC免疫表型CD1a^＋／CD40^＋、CD83^＋／CD86^＋、CD14^＋／HLA-DR^＋比例显著增高（57．6％±5．4％对33．2％±6．0％、32．5％±3．9％对26．0％±2．8％、38．1％±2．6％对29．1％±2．1％,P〈0．01）;IFN-γ含量呈时间依赖性增高;CTL细胞对HepG2细胞的杀伤作用显著增强（43.3％±11.3％对13．9％±4．6％,P〈0．01）。结论：应用HepG2细胞抗原孵育脐血CD34^＋造血干细胞可诱导分化成熟DC,DC可促进异基因淋巴细胞活化分泌IFN-γ,并产生特异性CTL细胞,杀伤肝癌HepG2细胞。     

功能性消化不良患者胃粘膜肥大细胞的增加

目的:探讨胃粘膜肥大细胞(MC)与功能性消化不良(FD)之间的关系。方法:对15例健康自愿者和34例FD患者进行研究。每例胃镜下取胃窦粘膜2块,用免疫组化方法染色MC并计数。结果:34例FD患者胃粘膜MC数明显多于健康对照者(19.48/HSP±3.14/HSP与16.07/HSP±2.13/HSP,P<0.05),其中12例(35.3%)FD患者MC数超过正常人上限。且MC数与幽门螺杆菌( H.pylori)。胃粘膜炎症有明显关系。结论:FD患者胃粘膜MC数增多,可能参与了FD的发病。     

Mononuclear cells are not involved in BGP synthesis and secretion

Summary Osteocalcin or bone GLA protein (BGP) is found at high levels in only two tissues, the extracellular matrix of bone and dentine. Tissue culture experiments have demonstrated that BGP is synthesized by two osteoblastic osteosarcoma cell lines (ROS 2/3 and 17/2) and by normal osteoblastic cells in primary culture. BGP was not found in rat cartilage nor in liver, kidney, lung, spleen, brain, heart, thymus, skeletal muscle. In this study secretion of BGP was assayed by RIA in the supernatants of 48-hour cultures of peripheral blood lymphocytes or monocytes. Lymphocyte cultures were carried out using RPMI-1640 supplemented with L-glutamine and antibiotics at the concentration of 1×10⁶ cells/ml and activated by PHA (10 ng/ml). Peripheral blood monocytes were purified by adherence to plastic Petri dishes and treated with cold PBS supplemented with EDTA. Monocytes were cultured as previously described and stimulated with LPS (50 ug/ml). Cell-free supernatants were obtained by centrifugation and stored at –20°C, until the BGP assay was performed. The authors did not observe secretion of detectable amounts of BGP in the supernatants of short-term lymphocyte or monocyte cultures. These data indicate that circulating mononuclear cells are not involved in BGP synthesis and secretion.     

Postbinge Effects of Acute Alcohol Intoxication on Hepatic Free Radical Formation

The present studies were performed to test the hypothesis that Kupffer and endothelial cells are activated after recovery from an acute alcohol binge, which is accompanied by formation of oxygen-derived radicals. These radicals have been implicated in the pathogenesis of alcohol-mediated tissue injury in a number of organs. Male Sprague-Dawley rats received an intravenous injection of 20% ethanol in saline (1.75 g/kg), followed by an intravenous infusion (250 to 300 mg/kg/hr) for 12 hr. At the end of 12-hr infusion, ethanol was replaced by saline, and the infusion was continued for a further 6 hr. This was referred to as the recovery period. The 6-hr recovery period was selected because superoxide anion generation by the perfused liver peaked at this time point. Superoxide anion formation by the perfused liver was measured by the superoxide dismutase-inhibit-able reduction of ferricytochrome c. Kupffer and endothelial cells were isolated for the determination of in vivo glucose uptake and in vitro superoxide anion release. Results show that a significant ( p < 0.05) amount of superoxide (1.54 nmol/min/g) was generated by the perfused liver at 6 hr recovery after 12 hr of ethanol infusion. Serum ALT activity was also elevated in this treatment group. Time-matched control-saline infused animals or ethanol-treated animals without a recovery period released <0.2 nmol/min/g of superoxide. The postrecovery superoxide production and an accompanying increase in the in vivo glucose uptake were also observed in isolated Kupffer and endothelial cells. Depletion of Kupffer cells by gadolinium chloride before ethanol treatment and recovery was associated with significant attenuation of free radical formation by the perfused liver and reduction of serum ALT. These studies demonstrate that recovery from an acute alcohol binge has a stimulating effect on hepatic sinusoidal superoxide production, and it may also affect liver function.