Lactobacilli Modulate Proliferation and Cytokine Production of Human Peripheral Blood Natural Killer Cells In Vitro

The intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. Evidence indicates that lactic acid bacteria (LAB), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. Established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. The objective of this study is to evaluate the immunomodulating properties of a range of LAB of human origin. As dendritic cells (DCs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro -generated immature DCs serve as a suitable model for studying the immunomodulating effects of lab. Human immature DCs were generated in vitro from monocytes and exposed to lethally UV-irradiated LAB. The effect of various species of LAB on DCs in direct contact was evaluated. Furthermore, the maturation pattern of DCs separated from the bacteria by an epithelial cell layer (CaCo-2 cells), which should mimic the intestinal environment, was studied. Cytokine secretion (IL-12, IL-10 and TNF-α) and upregulation of maturation surface markers on DCs (CD83 and CD86) was measured. Different LAB induced diverse cytokine responses. Some strains were strong IL-12 and TNF-α inducers and others weak. All strains induced IL-10. Different LAB also differentially modulated expression of CD83 and CD86 on DCs. Although some variation in the response to LAB of DCs generated from different blood donors was observed, general differences in the effect of the various LAB was revealed. Experiments with the DC CaCo-2 coculture system are ongoing. Different species of LAB differentially affect DC maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response.     

In Vitro Production of Monoclonal and Polyclonal Immunoglobulins by Peripheral Blood Mononuclear Cells in Human Plasma Cell Myeloma

The in vitro monoclonal and polyclonal immunoglobulin (Ig) production of peripheral blood mononuclcar cells was studied in human multiple myeloma (four IgG myelomas, one IgA myeloma) and in one patient with benign monoclonal gammopathy. Using an enzyme-linked immunosorbent assay with anti-class-specific antisera and antisera against idiotypic structures on the myeloma protein, it was possible to quantilate separately monoclonal and polyctonal Ig of the same class in cell culture supernatants. After stimulation with pokcweed mitogen (PWM) patients' cells produced lower amounts of polyclonal Ig than cells from healthy adults. In contrast, production of monoclonal Ig could not be enhanced by PWM. Moreover, the kinetics of monoclonal Ig production was different from that of polyclonal Ig. Myeloma cells contained large amounts of monoclonal Ig while their content of polyclonal Ig was low. A rapid release of preformed monoclonal Ig during the first day of culture was not inhibited by puromycin. A later phase of release was partly suppressed by puromycin and was probably caused by active protein synthesis.     

Hyperalgesia and Edema Responses Induced by Rat Peripheral Blood Mononuclear Cells Incubated with Carrageenin

The aim of this study was to verify the role played by mononuclear cells in an acute (nonimmune) inflammatory reaction. Mononuclear cells purified from rat peripheral blood were incubated for 1, 2, or 24 h with 100 or 250 g/ml carrageenin (Cg). The resultant donor supernatant was injected into recipient rats to test its ability to induce hyperalgesia (reduction in threshold for paw pressure) and edema (increase in paw volume). Mononuclear cell supernatants (MnS) induced a significant time- and dose-dependent hyperalgesia and edema in rat paws, which reached a maximal effect at 3 h, lasted for 6 h, and returned to basal levels at 24 h of injection. Prostaglandins and cytokines (interleukin 1, 2, 6, 8, and tumor necrosis factor alpha) accounted for the hyperalgesia induced by MnS, as it was reduced (40 to 90%) by synthesis inhibitors such as indomethacin, dexamethasone, rolipram, and cyclosporin added to the cultures at a microgram dose-range. Edema was dependent on serotonin release in rat paws. These results indicate that mononuclear cells may be important contributors to acute inflammatory reactions, especially under those conditions where pain is an important component.     

Nigerooligosaccharides Augments Mitogen-Induced Proliferation and Suppresses Activation-Induced Apoptosis of Human Peripheral Blood Mononuclear Cells

Nigerooligosaccharides (NOS), a mixture of nigerose and nigerosylmaltooligosaccharides, consists of immunopotentiating oligosaccharides found in foodstuffs. We have previously reported that activation of peripheral blood mononuclear cells (PBMC) in response to concanavalin A (Con A) or a streptococcal preparation of OK-432 is augmented in healthy young adults and elderly subjects after the intake of NOS-supplemented syrup. A reappraisal of the data suggests that NOS augments proliferation but partly suppresses activation-induced apoptosis of PBMC in response to these mitogens. To confirm this hypothesis, PBMC from healthy male subjects were stimulated with Con A or OK-432 in the presence of nigerose at the concentrations at which it was detected in the blood of subjects who had ingested NOS-supplemented syrup. Cellular activation, specifically metabolic demand, viability and proliferation, was assessed from glucose consumption, by WST-1 colorimetry and by 5-bromo-2'-deoxy-uridine incorporation assay, respectively. The Con A-induced activation of PBMC in each measurement was significantly augmented by nigerose. OK-432-induced decreases in the viability of PBMC were significantly inhibited by nigerose. Stimulation of PBMC with Con A or OK-432 induced apoptosis, but nigerose suppressed such activation-induced cell death. These results indicated that nigerose activated PBMC in vitro in a manner similar to the process observed in vivo, providing further evidence for the effectiveness of consumption of NOS-supplemented syrup.     

PPD对人内皮细胞与外周血单个核细胞粘附的影响

为探讨热休克蛋白 6 0kD在动脉粥样硬化早期形成机制中的作用 ,本实验采用病原微生物同源性蛋白即结核杆菌纯化蛋白衍生物 (PPD ) ,观察其在人脐静脉内皮细胞 (EC )和外周血单个核细胞 (PBMC )粘附过程中的作用。利用培养的人脐静脉内皮细胞株 ,测定人PBMC和EC的粘附率 ;采用McAbCD5 4和McAbIL 1β抑制试验 ,检测PPD对EC表达粘附分子的作用。结果显示 ,经PPD处理的EC对PBMC的粘附率增加 ,并有一定的时相关系 ,粘附分子ICAM 1在EC表面表达增加 ;McAbCD5 4可以降低PPD的EC和PBMC的粘附率 ,而McAbIL 1β对粘附率没有影响。PPD可以直接刺激EC表达粘附分子CD5 4,进一步参与介导白细胞与内皮细胞的粘附。     

Cytokine Production in Cell Culture by Peripheral Blood Mononuclear Cells from Immunocompetent Hosts

The production of interleukin 2 (IL-2) gamma interferon, IL-4, tumor necrosis factor alpha (TNF-α), TNF-β, IL-5, and IL-10 in vitro by peripheral blood mononuclear cells cultured from healthy immunocompetent subjects after mitogen stimulation was determined. The mitogens used were concanavalin A, phytohemagglutinin, pokeweed mitogen, and Staphylococcus aureus Cowen. The results obtained provide a normal range for the production of these cytokines under specified conditions in vitro.     

Ag85B表位多肽对体外单核细胞增殖影响的研究

目的通过观察Ag85B不同表位多肽对人外周血单个核细胞（PBMC）增殖的影响,筛选出增殖活性较强的Ag85B表位多肽,为制备结核新疫苗提供理论基础。方法采用的实验方法为人工预测Ag85B的表位,并合成多肽。将不同表位多肽体外刺激人外周血单个核细胞,利用CCK-8法测定单核细胞同一时相的吸光值,计算各自的增殖指数,并与BCG、Ag85B抗原等作对比。结果①Ag85B表位多肽组的OD450值及增殖指数均高于阴性对照组,且高于传统抗原BCG;②5条多肽中,28aa多肽的OD450值及增殖指数最高,且显著高于BCG（POD450=0.001〈0.01,P增殖指数=0.013〈0.05）,但低于PHA;③28aa多肽的OD450值及增殖指数明显低于Ag85B,差异有统计学意义（POD450=P增殖指数=0.000〈0.01）。结论预测的Ag85B表位多肽抗原具有一定的免疫原性,其中28aa多肽可作为优势表位多肽,但是否可用于表位多肽疫苗候选抗原还需进一步研究。     

低剂量辐射对外周血单个核细胞体外抗肿瘤活性的影响

目的：观察低剂量γ射线辐照的外周血单个核细胞对体外培养人胃癌细胞系MKN-28细胞的杀伤作用的影响。方法：实验设MKN-28肿瘤细胞对照组、外周血单个核细胞对照组、照射和未照射的外周血单个核细胞与肿瘤细胞共培养组，照射组的照射剂量为1Gy。利用吖啶橙／溴化乙锭（AO／EB）荧光双染色法，定期观察外周血单个核细胞对肿瘤细胞的杀伤情况。结果：外周血单个核细胞照射后培养至144h时有大量细胞死亡，存活细胞明显少于未照射组，培养至240h时．照射与非照射组中存活的外周血单个核细胞均减少，但是在照射组中减少更明显一些，在外周血单个核细胞与MKN-28肿瘤细胞共培养组中，照射组在共培养96h-240h时，外周血单个核细胞对胃癌细胞的杀伤作用比未照射组明显增强。结论：γ射线辐照对某些外周血单个核细胞有损伤作用，低剂量辐射的外周血单个核细胞杀伤肿瘤细胞活性增强，但肿瘤细胞对辐照后的外周血单个核细胞有趋化及细胞毒作用。     

外周血单核细胞SOCS-1表达与MODS患者预后关系的研究

目的了解外周血单核细胞SOCS-1表达与多器官功能障碍综合征（MODS）患者预后的关系及临床意义。方法收集24例MODS患者，并采集其外周静脉血，采用淋巴细胞分离液密度梯度离心法分离外周血单核细胞（PBMCs），分别以RT-PCR法及Western-blot法检测PBMCs中SOCS-1的基因及蛋白表达，分析其与预后及MODS评分的关系。结果MODS组中死亡患者PBMCs中的SOCS-1mRNA表达量（0.4938±0.0273）显著低于存活患者（0.5475±0.0289）（P〈0.05），SOCS-1蛋白表达量（0.7924±0.0284）显著低于存活患者（0.8406±0.0407）（P〈0.05）。MODS患者的PBMCs中的SOCS-1mRNA表达量与MODS评分呈显著的负相关关系（r=-0.723,P〈0.01），SOCS-1蛋白表达量与MODS评分呈显著的负相关关系（r=-0.534，P〈0.01）。结论在MODS中，SOCS-1的表达可能起到保护组织避免损伤的作用,SOCS-1表达的减少可能提示患者的预后不良     

In Vitro Immunoglobulin Production by Peripheral Blood Mononuclear Cells from Multiple Myeloma Patients and Patients with Benign Monoclonal Gammopathy

Peripheral blood mononuclear cells (PBM) from four patients with IgG myeloma and four patients with benign monoclonal gammopathy (BMG) were stimulated with pokeweed mitogen (PWM), and the in vitro immunoglobulin production over 7 days was measured with an enzyme-linked immunosorbent assay. All myeloma patients were sufficiently treated with cytostatic drugs. Their PBM did not produce monoclonal Ig in vitro, as opposed to PBM from two patients with BMG. Unseparated PBM from myeloma patients produced smaller amounts of polyclonal Ig than unseparated cells from normal donors. However, macrophage-depleted PBM from myeloma patients produced amounts of Ig comparable to those of normal donors when autologous or allogeneic adherent cells were returned in defined numbers. T cells from three of the four myeloma patients could provide help for the Ig production by B cells from healthy donors. These results indicate that functionally normal polyclonal B cells circulate in the blood of myeloma patients. The circulating T-cell population also has no obvious defect. In contrast, blood macrophages seemed to be altered with respect to their regulating function for polyclonal Ig production. The results obtained by using cell populations from patients with BMG did not differ from those of healthy people.     

The Effects of Fructus Xanthii Extract on Cytokine Release from Human Mast Cell Line (HMC-1) and Peripheral Blood Mononuclear Cells

In the present study, human mast cell line (HMC-1) and peripheral blood mononuclear cells (PBMNC) were pre-incubated with various concentrations of Fructus xanthii extract solution followed by being stimulated with phorbol myristate acetate and further incubated for 24 hours. The cytokines, in cell culture supernatants, of interleukin (IL)-4, IL-6, IL-8, GM-CSF, and TNF-α in the supernatant were measured by enzyme-linked immunosorbent assay. The results demonstrated that Fructus Xanthii could modulate the mast cell-mediated and PBMNC-mediated inflammatory and immunological reactions modulated by cytokines. And our study supplied the evidence of the mechanisms of Fructus Xanthii in treating inflammatory and allergic diseases.     

The Effect of Human Umbilical Cord Blood Cells on Survival and Cytokine Production by Post-Ischemic Astrocytes in Vitro

Cerebral ischemia induces death of all neural cell types within the region affected by the loss of blood flow. We have shown that administering human umbilical cord blood cells after a middle cerebral artery occlusion in rats significantly reduces infarct size, presumably by rescuing cells within the penumbra. In this study we examined whether the cord blood cells enhanced astrocyte survival in an in vitro model of hypoxia with reduced glucose availability. Primary astrocyte cultures were incubated for 2 h in no oxygen (95% N, 5% CO₂) and low glucose (1% compared to 4.5%) media. Cord blood mononuclear cells were added to half the cultures at the beginning of hypoxia. Astrocyte viability was determined using fluorescein diacetate/propidium iodide (FDA/PI) labeling and cytokine production by the astrocytes measured using ELISA. In some studies, T cells, B cells or monocytes/macrophages isolated from the cord blood mononuclear fraction with magnetic antibody cell sorting (MACS) were used instead to determine which cellular component of the cord blood mononuclear fraction was responsible for the observed effects. Co-culturing mononuclear cord blood cells with astrocytes during hypoxia stimulated production of IL-6 and IL-10 during hypoxia. The cord blood T cells decreased survival of the astrocytes after hypoxia but had no effect on the examined cytokines. Our data demonstrate that the tested cord blood fractions do not enhance astrocyte survival when delivered individually, suggesting there is either another cellular component that is neuroprotective or an interaction of all the cells is essential for protection.     

Analysis of CD28 and bcl-2 Expression on Peripheral Blood and Liver-Infiltrating Mononuclear Cells in Patients with Autoimmune Hepatitis

Because the underlying mechanism of hepatocellular damages in autoimmune hepatitis (AIH) still remains unclear, analysis of CD28 and bcl-2 molecules, which are critical for T cell activation and survival, was performed in patients with AIH. The number of CD28(+)CD4(+) peripheral blood mononuclear cells (PBMC) in corticosteroid (CS)-treated patients was comparable to normal control individuals but decreased in untreated AIH patients. In contrast, the number of CD28(+)CD8(+) PBMC was decreased in both CS-treated and untreated AIH patients. Analysis of liver-infiltrating mononuclear cells (LIMC) showed that the number of CD28(+)CD4(+) and CD28(−)CD8(+) LIMC were positively correlated with the histology activity index score. Bcl-2(+)CD4(+) LIMC were observed in the portal area of the liver and the numbers fluctuated with disease activity during the time course after CS administration. By contrast, CD8(+) LIMC were shown not to express bcl-2. Taken collectively, these results suggest that bcl-2(+)CD28(+)CD4(+) and bcl-2(−)CD28(−)CD8(+) cells may play critical and distinct roles in hepatocellular damage in AIH.     

人外周血树状突细胞在体外对LAK杀伤活性的影响

本文采用三因素不同水平的析因设计，研究了人外周血树状突细胞在体外对ＬＡＫ抗肿瘤作用的影响。结果显示，３种浓度的树状突细胞均能增强ＬＡＫ的杀伤活性（Ｐ＜０．００１），以中等浓度的树状突细胞作用最为明显。加入ＩＬ－２后，树状突细胞对ＬＡＫ抗肿瘤活性的增强程度进一步提高。     

A Cryopreservation Method of Human Peripheral Blood Mononuclear Cells for Efficient Production of Dendritic Cells

The establishment of a cryopreservation method for unstimulated fresh peripheral blood mononuclear cells (PBMC) with nearly 100% viability would greatly contribute to the conduct of various immunological experiments. The cells most sensitive to freezing and thawing procedure seem to be dendritic cells (DC) and their precursors, which are of the most potent antigen-presenting cells. The authors investigated and established a method of cryopreserving fresh PBMC from which DC were recovered and differentiated efficiently by using recombinant (r) GM-CSF and rIL-4. PBMC frozen in the presence of 12% dimethylsulfoxide and 25–30% fetal calf serum recovered DC as efficiently as freshly obtained PBMC. Established DC could also be cryopreserved in the presence of 12% DMSO with their viability maintained at more than 90%. The 12% DMSO freezing solutions were superior to both the 10% DMSO solution and the previously reported DC freezing medium (2 m or 15.4% DMSO). The DC obtained from the cryopreserved PBMC expressed HLA-DR, HLA-DQ, CD80 and CD86 antigens, and stimulated allogenic PBMC to an extent almost identical to that obtained from fresh PBMC. These findings indicate that the conditioned medium utilized here enables safe cryopreservation of DC and DC precursors in PBMC.     

Short-Chain Fatty Acids Regulate Cytokines and Th17/Treg Cells in Human Peripheral Blood Mononuclear Cells in vitro

Short-chain fatty acids (SCFAs) have been recognized as mediators of immune responses, including pathways of cytokine production. In this study, we investigated the immune-regulatory effects of SCFAs on human peripheral blood mononuclear cells (PBMCs) from buffy coat of healthy donors. PBMCs were exposed to varying concentrations of individual SCFAs or of their mixtures of acetate, propionate and butyrate. The productions of interleukin (IL) IL-1β, IL-2, IL-6, IL-10, IL-17, IL-21, IL-23 and transforming growth factor beta 1 (TGF-β1) were assessed. T cell differentiation after exposure to SCFAs was also examined. Compared with lipopolysaccharide (LPS)-stimulated cells (controls), SCFAs slightly decreased TGF-β1 production and reduced IL-6 production; butyrate was more effective than acetate or propionate. SCFAs particularly butyrate caused the induction of CD4+CD25+ regulatory T cells (Treg) rather than Th17 cells. SCFAs may up-regulate the production of anti-inflammatory cytokines in PBMCs, resulting in the induction of CD4+CD25+ Treg cells.     

Mycobacterium Tuberculosis Beijing Genotype Induces Differential Cytokine Production by Peripheral Blood Mononuclear Cells of Healthy BCG Vaccinated Individuals

Members of the Mycobacterium tuberculosis (Mtb) Beijing genotype are a major concern due to their high prevalence in tuberculosis patients and their high rate of multi-drug resistance. Although it has been shown that Beijing modifies macrophage behavior, little is known about how this genotype could affect the cellular immune response. In order to address this issue, peripheral blood mononuclear cells (PBMC) from healthy BCG vaccinated individuals were stimulated with protein extracts from three Mycobacterium tuberculosis genotypes: Canetti, H37Rv and Beijing evaluating T cell proliferation and cytokine production. In this system both CD4+ and CD8+ proliferated in a similar manner independently of the Mtb genotype used for stimulation. Regarding cytokines, all strains induced similar levels of IFN-γ, but were unable to induce IL-4 and TGF-β. Contrasting, Canetti strain induced lower production of IL-10, TNF-α and IL-12 compared to H37Rv and Beijing. Interestingly, PBMC stimulated with the Beijing strain produced the highest levels of IL-12 and IL-10 than those stimulated with other strains. This differential cytokine expression could affect the pathogenesis induced by Beijing strain through the modulation of inflammatory process in the host, but the precise mechanisms by which this cytokine environment affects the Beijing strain pathogenesis needs further characterization.     

新城鸡瘟病毒诱导人粘附性单个核细胞产生一氧化氮与细胞毒性

本文采用Griess试剂、间接免疫荧光法和同位素释放等方法,发现新城鸡瘟病毒Losota系（NDV-L）与人外周血粘附性单个核细胞（a-MCs）作用2～4h后,可稳定地吸附在a-PBMCs表面,并使其释放一氧化氮（NO）,释放量与阳性对照组（BCG-LPS作用的a-PBMCs）相近;采用~3H-TdR释放法测定NDV-L作用的a-PBMCs对K_(562)靶细胞的细胞毒活性,发现具有明显的杀伤效应,且该杀伤效应与NO的产生有一定的依赖性。     

MHC-Unrestricted Lysis of MUC1-Expressing Cells by Human Peripheral Blood Mononuclear Cells

Many human adenocarcinomas can be killed in vitro by targeted cytotoxic T-lymphocytes (CTL); however, major histocompatibility complex (MHC)-restrictions are typically required. The MUC1 antigen is common in many human adenocarcinomas, and is associated with a variable number of tandem repeats. It has been proposed that antigens with such repeated epitopes may be vulnerable to cytotoxic T-lymphocyte killing without MHC-restriction. Therefore, it is possible that MUC1-expressing malignant cells may be killed by targeted cytotoxic T-lymphocyte in the absence of MHC-restriction. In this study, a human MUC1-expressing murine mammary carcinoma cell line was used to determine if cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells requires MHC-restriction. Specifically, MUC1-stimulated human mononuclear cells (M1SMC) were observed to kill human MUC1-transfected, MUC1-expressing murine mammary carcinoma cells, but not the mock-transfected, non-MUC1-expressing murine mammary carcinoma cells. Furthermore, the killing was blocked by antibody to MUC1, indicating MUC1-specific killing. In conclusion, cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells can be MHC-unrestricted.     

The Effect of Disodium Cromoglycate on In Vitro Proliferation of Peripheral Blood Mononuclear Cells from Allergic and Healthy Donors

The effect of disodium cromoglycate on in vitro proliferative responses of peripheral blood mononuclear cells from healthy individuals, allergic patients with moderate serum IgE and patients with atopic dermatitis and high levels of serum IgE was investigated. Peripheral blood mononuclear cells were stimulated with mitogens (phytohaemagglutinin, Concanavalin A), recombinant interleukin-2, calcium ionophore + phorbol 12-myristate 13-acetate, purified protein derivative of tuberculin and allergens. It was possible to induce in vitro specific, allergen-triggered responses only in allergic individuals with moderate serum IgE and not in individuals with atopic dermatitis and high serum IgE. Generally, whenever the stimulatory signal(s) caused a significant proliferative response, disodium cromoglycate inhibited the proliferation. This inhibition was seen for all activation agents and for both healthy and allergic individuals. By contrast, for certain non- or low-responders (both healthy and allergic individuals) disodium cromoglycate seemed to amplify the proliferation to various activation signals. Only non- or low-responder cells derived from atopic dermatitis patients showed a biphasic kinetic response pattern when stimulated with the drug in combination with recombinant interleukin-2, recombinant interleukin-2 + ionophore or specific allergens.