Characteristics of Chicken Intraembryonic Cells that Express B-L (Ia-Like) Antigen under the Influence of Cultured Bursal Epithelium

In-vitro-cultured bursal epithelium (BE) and BE-conditioned medium (BECM) induce B-L antigen on chicken intraembryonic cells. Cells prepared from 9-day-old intraembryonic mesoderm were fractionated in accordance with cell size by linear albumin gradient sedimentation at 1 g. Two cell types could be distinguished on which expression of the B-L antigen was altered after a 6-h incubation with the bursal epithelial component. One fraction contained small mononuclear cells with low sedimentation velocity (≤ 3 mm/h) and low spontaneous proliferation activity. These cells responded strongly to BECM and showed a slight but not significant response to BE (index after incubation with BECM 3.8, with BE 1.7, as compared with RPMI medium control). The other fraction was composed of large mononuclear cells with sedimentation velocity > 9 mm/h and with high spontaneous proliferation. These cells showed a response of equal magnitude to both BE and BECM (index after incubation with BE 2.0, with BECM 2.3). These results suggest that the bursa of Fabricius has influence on two different cell types: a large, probably primitive undifferentiated cell, responding equally to bursal cellular contacts and BE culture supernatant, and a small mononuclear cell type, probably more mature, responding more clearly to the bursal humoral factor.     

B-L Antigens (Ia-like Antigens) of the Chicken Major Histocompatibility Complex

This paper reviews the present knowledge of B-L antigens encoded by the chicken B complex as regards to the following aspects: (1) identification and cellular expression, (2) structural studies, (3) evidence for two distinct populations of B-L antigens, (4) mapping of B-L loci of the B complex, (5) B-L and immune response, and (6) the role of the B-L antigens for the control of mixed lymphocyte reactions (MLR) and graft-versus-host (GVH) reactions. It is concluded that B-L antigens of the chicken exhibit extensive homology with mammalian la antigens. A genetic map of the B complex is presented.     

慢性乙型肝炎患者外周血来源树突状细胞的功能状态

通过比较细胞因子诱导的外周血来源的慢性乙型肝炎患者树突状细胞 (DC )和正常人DC在免疫分子表达和免疫功能等方面的差别 ,探讨慢性乙型肝炎患者DC所处的功能状态。以细胞计数、间接荧光表型分析、MTT法测定DC对同种异体淋巴细胞的刺激作用、ELISA法测定DC分泌IL 1 2、IL 6等方法进行研究。实验结果表明 :慢性乙型肝炎患者相同数量的前体细胞经诱导分化形成的DC数量明显减少 ;在培养的 3、 6、 9d,与同期正常人相比 ,慢性乙型肝炎患者的DC表达CD1、CD83、CD80和HLA DR分子水平均较低 ,而CD1 4分子表达水平较高 ;刺激同种异体淋巴细胞增殖能力也低于同期正常人 ;在培养的第 6天DC分泌的上清液中 ,慢性乙型肝炎患者DC分泌的IL 1 2水平低于正常人 ,而分泌的IL 6水平高于正常人。结果提示 ,慢性乙肝患者外周血来源的DC免疫功能处于抑制状态 ,细胞因子表达异常。     

Functional Property of la-Positive T Cells in Peripheral Blood from Patients with Systemic Lupus Erythematosus

Ia-positive (Ia+) T cells in peripheral blood and their functional property were examined in patients with systemic lupus erythematosus (SLE). Binding of specific monoclonal antibodies was assessed by indirect immunofluorescence. Functional study of Ia+ T cells was carried out in coculture experiments by measuring the IgG secreted into the culture supernatant. We found that the percentage of Ia+ T cells in peripheral blood from patients with SLE was raised and the rise correlated positively with serum gamma globulin and IgG level. The elevation was further increased after stimulation with DNA in vitro, indicating the presence of DNA-sensitive T cells. Functionally, Ia+ T cells acted as helper cells in spontaneous IgG synthesis of SLE B cells, and were enriched in the OKT4 subset. These results indicate that SLE T cells are activated in vivo and that the Ia+ T cells may play a crucial role in the immunoregulatory function. Accordingly, demonstration of Ia antigens on T cells by monoclonal antibody may provide a useful tool for the measurement of immunological activity in patients with SLE.     

不同方法分离外周血单个核细胞对树突状细胞增殖的影响

目的 :探讨不同方法分离的外周血单个核细胞 (PBMC)对树突状细胞 (dentriticcells,DC)增殖产量的影响。方法 :取肝素抗凝血 ,分别用羟乙基淀粉 (hydroxyethy1starch ,HES)离心沉淀法、Ficoll密度梯度离心法分离单个核细胞 ,直接进行贴壁计数及DC的诱导培养。结果 :HES离心沉淀法、Ficoll密度梯度离心法分离的外周血单个核细胞 ,其贴壁细胞经FACS检测 ,CD1 4 细胞分别占贴壁总数的 6 5 0 6 %、6 3 80 % ,每 4 0ml外周血扩增的DC产量分别为 (0 .88～ 3.1 7)× 1 0 6和 (0 .79～ 3.0 2 )× 1 0 6。结论 :羟乙基淀粉法分离的PBMC更适用于DC的诱导培养。     

Expression and Functional Analysis of Toll-Like Receptors of Peripheral Blood Cells in Asthmatic Patients: Implication for Immunopathological Mechanism in Asthma

Background  We investigated the expression profile of toll-like receptors (TLRs) and TLR ligand-activated production profile of asthma-related inflammatory cytokines in asthmatic patients. The expression of TLR1–8 on monocytes, CD4+ T helper lymphocytes, CD8+ T cytotoxic lymphocytes, CD19+ B lymphocytes, and dendritic cells, and ex vivo production of cytokines from peripheral blood mononuclear cells activated by TLR ligands were measured by flow cytometry. Discussion  Ex vivo productions of TNF-α, IL-10, and IL-1β by TLR4 and TLR5 ligand LPS and flagellin were significantly lower in asthmatic patients (all P < 0.05). Expression of TLR4 and TLR5 was also found to be significantly lower in asthmatic patients when compared to that of control subjects (all P < 0.05). Therefore, the decreased activation of TLR4 and TLR5 in asthmatic patients might contribute to the immunopathological mechanisms of asthma by reducing the release of Th1 and anti-inflammatory cytokines. Samantha W. M. Lun and C. K. Wong contributed equally to this study.     

Optimization and Limitations of Use of Cryopreserved Peripheral Blood Mononuclear Cells for Functional and Phenotypic T-Cell Characterization

The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4⁺ and CD8⁺ T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at −70°C for ≤3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at −70°C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.Utilization of cryopreserved peripheral blood mononuclear cells (PBMC) for immunologic assays has dramatically increased in recent years. Cryopreserved PBMC are particularly useful in clinical trials with low end-point frequencies because they allow the immunologic assays to be performed after the conclusion of the studies, when all the end points have already been identified (1, 14, 18). In addition, the use of cryopreserved PBMC allows all assays to be performed in a single laboratory, eliminating interlaboratory variability, which has been a confounder in some studies (15). Furthermore, if changes in immunologic parameters over time are the main outcome of the immunologic studies, interassay variability may become a confounder, too. Testing all the cryopreserved PBMC per subject at one time can eliminate this confounder.The use of cryopreserved PBMC in immunological assays poses challenges, including the availability of adequate equipment (7) and the need for technical proficiency. Assays have to be adapted and validated for the use of cryopreserved PBMC (4, 6, 9, 11, 17, 19), and the quality of the frozen cells has to be monitored (5) to ensure reliable results in functional and phenotypic assays.The Cryopreservation Working Group (WG) of the Pediatric AIDS Clinical Trials Group (PACTG), which operated between 1999 and 2006, developed a series of experiments aimed at optimizing methods of PBMC cryopreservation for assays requiring viable cells, adapting immunologic assays to cryopreserved PBMC, and establishing quality control parameters for immunologic assays with cryopreserved PBMC. The group focused on highly complex functional and phenotypic assays commonly used as outcome measures in studies that address immune suppression or reconstitution. Unlike previous studies of cryopreserved PBMC in immunologic assays, which were done at single laboratories (3, 8, 13, 17), our study presents results obtained at eight laboratories, substantiating the generalizibility of our findings.     

Human Epidermal Langerhans Cells and Peripheral Blood Monocytes

We compared the functional capacities of human epidermal Langerhans cells (LC) and peripheral blood monocytes. Epidermal sheets were obtained by a suction blister device. After enzymatic treatment LC were enriched by attaching them to IgG-located erythrocyte monolayers. On a per cell basis, LC were several times more efficient accessory cells than monocytes in augmenting nickel-and tuberculin (PPD)-induced T-cell proliferation. In mixed cell cultures LC stimulated both autologous and allogeneic T cells, whereas monocytes stimulated only allogeneic cells. In addition, LC were significantly more potent allogeneic stimulators than monocytes. Although monocytes were weaker accessory cells and allogeneic stimulators than LC, they induced higher interleukin 1 (IL-1) activities than LC-enriched or LC-depleted cells. These results indicate that there are functional differences between LC and monocytes and that antigen presentation and mediator secretion are not correlated.     

孕妇外周血液中胎儿红细胞的研究进展

研究认为孕妇外周血液循环中出现胎儿红细胞的主要原因是脐动脉和绒毛间隙之间的压力不同造成的。如果绒毛屏障受到破坏时,胎儿的血液就会通过绒毛间隙,进入到母体,最终出现在母体的外周血液循环中。通常可能会有很少量的胎儿红细胞在母体的外周血液循环中出现,这是正常的情况,且没有明显的胎儿和母体不良反应。但是当胎儿进入母体的血量超过一定阈值时,就会引发胎母输血综合征,引起严重的临床症候群。本文主要从胎儿红细胞的来源;现有检测方法;对胎母输血综合征的诊治;与新生儿贫血的相关性;在产前诊断中的价值;及对子痫前期发病预测等临床应用进展方面进行分析总结,为临床诊疗提供依据。     

Cytotoxic Activity of Peripheral Blood NK Cells towards Trophoblast Cells during Pregnancy

Bulletin of Experimental Biology and Medicine - We evaluated cytotoxic activity of peripheral blood NK cells towards trophoblast cells. NK cells either isolated or in the composition of mononuclear...     

Separation Methods of T Cells,Natural Killer,and Dendritic Cells from Peripheral Blood of Cancer Patients using Interleukin-2 and Functional Analysis of Natural Killer Cells after Separation

We aimed to induce three different immune cell subsets from a single blood sample from cancer patients to target different biological characters of cancer cells. In the presence of 6000 IU/ml IL-2, natural killer (NK) cells adhere to plastic. By using this ability, we could separate dendritic cells, T cells, and NK cells from peripheral blood mononuclear cells. The cultured NK cells demonstrated higher nonspecific cytotoxicity against tumor cell lines than did the T cells. Furthermore, adherent NK cells demonstrated higher cytotoxicity than nonadherent NK cells, although there was no difference between adherent and nonadherent NK cells in natural cytotoxicity receptors (NKp30, NKp44, NKp46) and NKG2D expression. With these results, we confirmed that we could induce dendritic cell, T cell, and higher cytotoxic NK cells from a single blood draw, and this methodology facilitates to the use of these cells for clinical grade conditions.     

Effect of Contaminating Red Blood Cells on OKT3-Mediated Polyclonal Activation of Peripheral Blood Mononuclear Cells

Erythrocytes are typically present as impurities in the majority of peripheral blood mononuclear cell (PBMC) preparations. This study was undertaken to investigate the effects of contaminating red blood cells (RBC) on the ability of OKT3 to activate CD4⁺ and CD8⁺ T cells. Surprisingly, the levels of gamma interferon, tumor necrosis factor alpha, and interleukin-1β (IL-1β) produced by PBMC upon stimulation by OKT3 were increased (P < 0.05) in a dose-dependent manner when increasing amounts of autologous RBC (RBC-to-PBMC ratios of 2:1, 10:1, and 50:1) were spiked into PBMC preparations. The OKT3-driven induction of the IL-2 receptor (CD25) and the proliferation of T lymphocytes in response to phorbol myristate acetate were not affected by the addition of RBC.     

Triggering Receptor in Myeloid Cells (TREM-1) Specific Expression in Peripheral Blood Mononuclear Cells of Sepsis Patients with Acute Cholangitis

To determine its relationship with acute cholangitis (AC), we sought to quantify expression of triggering receptor expressed on myeloid cells (TREM-1) in peripheral blood mononuclear cells (PBMC) of sepsis patients with AC. Peripheral blood samples of 42 AC patients and 48 patients with AC of severe type (ACST) were collected from January to September, 2008 and tested for TREM-1 mRNA by RT-PCR and protein expression by immunocytochemistry and Western blotting. ELISA and immunoturbidimetry were employed to detect the changes of TNF-α or C-reactive protein in the serum respectively. TREM-1 expression was higher in ACST group than in AC group (P < 0.01). TREM-1 was positive in mononuclear cells by immunochemistry in both groups before operative therapy, but the positive expression rate decreased at 48 h postoperatively. Compared with healthy controls, TREM-1 protein expression levels were up-regulated in sepsis patients with AC. TREM-1 expression has highly sensitivity and specificity in sepsis patients with AC or ACST. TREM-1 is up-regulated in PBMC of AC patients, and has higher sensitivity and specificity than other clinical inflammation markers, suggesting its importance in AC-induced sepsis.     

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.     

Osteoclastogenic Potential of Peripheral Blood Mononuclear Cells in Cleidocranial Dysplasia

Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal dysplasia characterized by hypoplastic or aplastic clavicles, dental abnormalities, and delayed closure of the cranial sutures. In addition, mid-face hypoplasia, short stature, skeletal anomalies and osteoporosis are common. We aimed to evaluate osteoclastogenesis in a child (4 years old), who presented with clinical signs of CCD and who have been diagnosed as affected by deletion of RUNX2, master gene in osteoblast differentiation, but also affecting T cell development and indirectly osteoclastogenesis. The results of this study may help to understand whether in this disease is present an alteration in the bone-resorptive cells, the osteoclasts (OCs). Unfractionated and T cell-depleted Peripheral Blood Mononuclear Cells (PBMCs) from patient were cultured in presence/absence of recombinant human M-CSF and RANKL. At the end of the culture period, OCs only developed following the addition of M-CSF and RANKL. Moreover, real-time PCR experiment showed that freshly isolated T cells expressed the osteoclastogenic cytokines (RANKL and TNFα) at very low level, as in controls. This is in accordance with results arising from flow cytometry experiments demonstrating an high percentage of circulating CD4⁺CD28⁺ and CD4⁺CD27⁺ T cells, not able to produce osteoclastogenic cytokines. Also RANKL, OPG and CTX serum levels in CCD patient are similar to controls, whereas QUS measurements showed an osteoporotic status (BTT-Z score -3.09) in the patient. In conclusions, our findings suggest that the heterozygous deletion of RUNX2 in this CCD patient did not alter the osteoclastogenic potential of PBMCs in vitro.     

In Situ Hybridization of Interferon-Gamma Producing Peripheral Blood Mononuclear Cells

Individual interferon-gamma (IFN-gamma) producing cells in activated human peripheral blood mononuclear cells (PBMC) were characterized by in situ hybridization using [35S]-labelled antisense RNA probes. The proportion of positive cells expressing IFN-gamma mRNA varied according to the substances used for stimulation. IFN-gamma mRNA expressed a relatively low percentage of 1-8% PBMC after a single stimulus with mitogens or OKT-3 antibody and 20-30% of the cells were identified to synthesize IFN-gamma mRNA after stimulation with PHA + P-MA + OKT-3 antibody. The expression of IFN-gamma mRNA and production of the lymphokine was dependent on accessory cells. If accessory cells were replaced by recombinant interleukin-1 (IL-1) plus interleukin-6 (IL-6), then T-cell proliferation to phytohaemagglutinin (PHA) could be partially restored and measurable amounts of IFN-gamma were detected. The addition of interleukin-2 (IL-2) or phorbol-12-myristate-13-acetate to T cells stimulated with PHA, IL-1 and IL-6 did not restore the production of IFN-gamma to an extent comparable to that produced by T cells stimulated in the presence of accessory cells. In further studies, depletion of T-cell subsets showed that CD3+, CD4+, CD8+, CD29+ and CD45RA+ cells were involved in IFN-gamma production after mitogenic stimulation. In conclusion, our data demonstrate that IFN-gamma production is dependent on signals from accessory cells and IFN-gamma is synthesized by only a small proportion of T cells, that did not belong to a unique population, characterized by conventional cellular surface antigens.     

某些肝病患者外周血单个核细胞天然杀伤(NK)活性的探讨

目前认为,NK细胞可能参与构成机体抗肿瘤生长和病毒感染的第一道防线。对许多恶性肿瘤患者的研究表明,其外周血NK活性低下,且与肿瘤进展和转移有关。但对病毒感染患者NK活性变化研究尚少,结果也不一致。一般认为,乙型病毒性肝炎和肝硬化(LC)可演变成肝细胞性肝癌(HCC)     

Immunomodulating Effects of CKBM on the Cytokine Production in Peripheral Blood Mononuclear Cells (PBMCs) from Healthy Volunteers

The current study investigated the immunomodulating effect of CKBM on cytokine induction in peripheral blood mononuclear cells (PBMCs) isolated from 20 healthy volunteers. Cytometric Bead Analysis (CBA) was used to study IL‐2, IL‐4, IL‐6, IL‐10, TNF‐alpha and IFN‐gamma. TNF‐alpha and IL‐6 were significantly increased in a CKBM dose‐ and time‐dependent manner. Flow cytometry analysis showed an increased intracellular staining of IL‐6 but not of TNF‐alpha in CKBM treated PBMCs. In addition, MTT cell cytotoxicity assay showed that CKBM concentrations below 5% did not significantly affect the metabolic activities of PBMCs. The current study indicated that CKBM may modulate the immune response by inducing the secretions of TNF‐alpha and IL‐6, which are cytokine mediators of innate immunity and inflammation preparing or “priming” the body to combat diseases.