从实验感染OPPV的山羊外周血单核细胞中分离病毒的研究

用绵羊胎肺细胞与接种绵羊进行性肺炎病毒（OPPV）的山羊外周血单核细胞共同培养的方法可以分离到病毒,这说明OPPV可以感染山羊。用细胞病变观察、间接荧光抗体试验、电镜切片观察和聚合酶链式反应对分离毒进行了鉴定,进一步证实了分离毒为OPPV。分离结果表明这是一种较为敏感的分离方法。绵羊胎肺细胞可传到40多代,且每一代次的细胞都可用于病毒的分离,因此这是一种非常实用的分离OPPV的方法。     

Identification of interleukin-13 related biomarkers using peripheral blood mononuclear cells

Asthma is a chronic disorder characterized by airway inflammation, reversible bronchial obstruction, hyper-responsiveness and remodelling. Data from human in vitro studies and experimental in vivo models of asthma has implicated interleukin (IL)-13 in the asthma phenotype suggesting that a therapeutic agent against it could be effective in treating asthma. The role of biomarkers is becoming increasingly important in the clinical development of therapeutics. Here we describe the use of the GeneChip^® DNA microarray technology platform to explore and identify potential response to therapy biomarkers that are associated with the biology of IL-13. Peripheral blood mononuclear cells (PBMCs) from eight healthy donors were cultured in the presence of IL-13, IL-4, an anti-IL-13 monoclonal antibody (mAb) or an isotype control mAb, and RNA from the treated cells was subjected to microarray analysis. The results revealed a number of genes, such as CCL17 (TARC), CCL22 (MDC), CCL23 (MPIF-1), CCL26 (eotaxin 3) and WNT5A (human wingless-type MMTV integration site family member 5A), that showed increased expression in the IL-13 and IL-4 treatment groups. Real-time polymerase chain reaction (PCR) subsequently confirmed these results. A follow-up study in PBMCs from five additional healthy donors showed that the neutralization of IL-13 completely blocked IL-13-induced TARC, MDC and eotaxin 3 production at the protein level. These data suggest that TARC, MDC, eotaxin 3, CCL23 and WNT5A if validated could serve as potential biomarkers for anti-IL-13 therapeutics.     

Identification of interleukin-13 related biomarkers using peripheral blood mononuclear cells

Asthma is a chronic disorder characterized by airway inflammation, reversible bronchial obstruction, hyper-responsiveness and remodelling. Data from human in vitro studies and experimental in vivo models of asthma has implicated interleukin (IL)-13 in the asthma phenotype suggesting that a therapeutic agent against it could be effective in treating asthma. The role of biomarkers is becoming increasingly important in the clinical development of therapeutics. Here we describe the use of the GeneChip^® DNA microarray technology platform to explore and identify potential response to therapy biomarkers that are associated with the biology of IL-13. Peripheral blood mononuclear cells (PBMCs) from eight healthy donors were cultured in the presence of IL-13, IL-4, an anti-IL-13 monoclonal antibody (mAb) or an isotype control mAb, and RNA from the treated cells was subjected to microarray analysis. The results revealed a number of genes, such as CCL17 (TARC), CCL22 (MDC), CCL23 (MPIF-1), CCL26 (eotaxin 3) and WNT5A (human wingless-type MMTV integration site family member 5A), that showed increased expression in the IL-13 and IL-4 treatment groups. Real-time polymerase chain reaction (PCR) subsequently confirmed these results. A follow-up study in PBMCs from five additional healthy donors showed that the neutralization of IL-13 completely blocked IL-13-induced TARC, MDC and eotaxin 3 production at the protein level. These data suggest that TARC, MDC, eotaxin 3, CCL23 and WNT5A if validated could serve as potential biomarkers for anti-IL-13 therapeutics.     

Proteomic map of peripheral blood mononuclear cells

In the field of proteomics extensive efforts have been focused on the knowledge of proteins expressed by different cell types. In particular, enormous progress has been done in the characterization of blood cellular components. In this work, we have established a public 2-DE database for human peripheral blood mononuclear cells (PBMCs) proteins. Two hundred and forty-six spots corresponding to 174 different proteins have been identified on 2-DE gels from PBMCs isolated from six healthy individuals. All the identified proteins have been classified in thirteen categories on the basis of their differential functions or cellular localization and annotated at the http://physiology.unile.it/proteomics. The role of several proteins has been discussed in relation to their biological function. We intend to show the potentiality of PBMCs to investigate the proteomics changes possibly associated with a large number of pathologies such as autoimmune, neurodegenerative and cancer diseases.     

In vitro immunization can elicit the expansion of diverse repertoire of B cells from peripheral blood mononuclear cells

We previously developed an in vitro immunization (IVI) protocol of human peripheral blood mononuclear cells (PBMC) for generating antigen-specific human antibodies. In order to clarify whether IVI protocolinduces antigen-specific B cell responses in PBMC, we analyzed family gene usage and sequence of the variable region gene of immunoglobulin heavy chain (VH gene) of the antibody produced from the in vitro immunized PBMC. Sequence homology analyses of VH gene demonstrated that a larger repertoire of B cells can be sensitized with mite-extract than with cholera toxin B subunit and rice allergen. Further, antigen-specific B cells were efficiently expanded by using CpG oligodeoxynucleotide as adjuvant. These results suggest that appropriate combination of sensitizing antigen and adjuvant is primarily important for expansion of antigen-specific B cells in IVI protocol.     

Differential expression of interferon-gamma,IL-4 and IL-10 in peripheral blood mononuclear cells during early pregnancy of the bovine

Maternal systemic immune response is regulated by conceptus-derived signals through peripheral blood mononuclear cells (PBMCs) via blood circulation during early pregnancy in cattle. In this study, the PBMCs from day 18 in non-pregnant cows and days 14, 18 and 30 in pregnant cows were used to explore the expression of interferon-gamma (IFN-γ), interleukin 4 (IL-4) and IL-10, and the plasma progesterone (P4) concentration was also determined. The results showed that the expression levels of mRNA and the protein of IFN-γ were lower and that IL-4 and IL-10 were higher in the PBMCs from the pregnant cows than in those of non-pregnant cows. From this study, early pregnancy induced a lower Th1 immunity (IFN-γ) and a higher Th2 immunity (IL-4 and IL-10) in the PBMCs, which may be related to interferon-tau and P4, thereby contributing to successful pregnancy in cattle.     

Cytokines profile and peripheral blood mononuclear cells morphology in Rett and autistic patients

A potential role for immune dysfunction in autism spectrum disorders (ASD) has been well established. However, immunological features of Rett syndrome (RTT), a genetic neurodevelopmental disorder closely related to autism, have not been well addressed yet.By using multiplex Luminex technology, a panel of 27 cytokines and chemokines was evaluated in serum from 10 RTT patients with confirmed diagnosis of MECP2 mutation (typical RTT), 12 children affected by classic autistic disorder and 8 control subjects. The cytokine/chemokine gene expression was assessed by real time PCR on mRNA of isolated peripheral blood mononuclear cells (PBMCs). Moreover, ultrastructural analysis of PBMCs was performed using transmission electron microscopy (TEM).Significantly higher serum levels of interleukin-8 (IL-8), IL-9, IL-13 were detected in RTT compared to control subjects, and IL-15 shows a trend toward the upregulation in RTT. In addition, IL-1β and VEGF were the only down-regulated cytokines in autistic patients with respect to RTT. No difference in cytokine/chemokine profile between autistic and control groups was detected. These data were also confirmed by ELISA real time PCR. At the ultrastructural level, the most severe morphological abnormalities were observed in mitochondria of both RTT and autistic PBMCs.In conclusion, our study shows a deregulated cytokine/chemokine profile together with morphologically altered immune cells in RTT. Such abnormalities were not quite as evident in autistic subjects. These findings indicate a possible role of immune dysfunction in RTT making the clinical features of this pathology related also to the immunology aspects, suggesting, therefore, novel possible therapeutic interventions for this disorder.     

母体外周血中胎儿有核红细胞的分离和富集方法的研究进展

母体外周血中分离的胎儿有核红细胞（fNRBCs）包含胎儿完整的遗传信息,可用于无创产前诊断。fNRBCs的分离和富集方法主要分为三类：物理分选法、抗原-抗体结合分离法和增殖法。不同的方法获得的fNRBCs的数量和纯度不同,多种方法联合使用可以提高富集产物中fNRBCs的纯度和数量。本文就母体外周血中fNRBCs的分离和富集方法进行综述。     

Effect of extremely-low-frequency pulsed magnetic fields on the mitogenic response of peripheral blood mononuclear cells

The effect of extremely-low-frequency pulsed magnetic fields (PMF) on the response of human peripheral blood mononuclear cells to mitogenic stimulation is reported. We investigated 25 healthy control subjects. Mitogen-stimulated mononuclear cells were exposed to PMF for 72 h and an inhibition of 3H-thymidine uptake was observed in all but one subject. The degree of inhibition of 3H-thymidine uptake was as much as 60%. There was no significant difference between the blastogenic responses of mononuclear cells exposed to PMF for 12 h and control cultures. This study establishes an inhibitory effect of PMF on an in vitro measure of immune function.     

Apoptosis of human peripheral blood mononuclear cells by wild‐type measles virus infection is induced by interaction of hemagglutinin protein and cellular receptor,SLAM via caspase‐dependent pathway

Although MV infection causes lymphopenia and degradation of cell‐mediated immunity, the mechanisms are poorly known. MV interacts with cellular receptors which mediate virus binding and uptake and are on the surface of PBMC. In this study, apoptosis of MV‐infected PBMC in vitro was analyzed. Both PBMC treated with UV‐inactivated viruses and those infected with live MV underwent apoptosis. Apoptosis of wild‐type MV‐infected PBMC was blocked by anti‐SLAM and anti‐MV hemagglutinin antibodies, respectively. Furthermore, addition of soluble MV hemagglutinin recombinant protein induced apoptosis in PBMC. These data suggest that induction of apoptosis in MV‐infected PBMC is triggered by interaction between hemagglutinin protein of MV and receptor, without other viral components. To further determine the mechanisms of apoptosis, caspase activity was analyzed by Western blotting. Wild‐type virus Yonekawa strain‐induced apoptosis was blocked by pretreatment with pan‐caspase inhibitor (Z‐VAD‐fmk). Intriguingly, the laboratory‐adapted Nagahata strain‐induced apoptosis was not blocked by Z‐VAD‐fmk, indicating that there may be different apoptosis pathways which depend on the viral receptors, SLAM and CD46. Both extrinsic and intrinsic apoptotic pathways, including activation of caspase‐3, ‐8 and ‐9, are involved in Yonekawa strain‐induced apoptosis. Taken together, the findings of this study could open up a new avenue for understanding the molecular mechanisms of MV‐induced PBMC apoptosis and immunosuppression.     

Taurine chloramine modulates cytokine production by human peripheral blood mononuclear cells

Summary.  The effect of taurine (Tau) and taurine chloramine (Tau-Cl) on the production of TNF-α, IL-1β, and IL-6 by peripheral blood mononuclear cells of healthy volunteers was examined. Cells were stimulated with bacterial lipopolysaccharide (LPS) in the presence of either Tau or Tau-Cl. After 24 h culture the cytokine concentrations were measured in both culture supernatants (secreted) and cell lysates (cell-associated) using ELISA. In LPS-stimulated cells Tau-Cl inhibited both the secreted and cell-associated IL-1β and IL-6, while exerted dual effect on TNF-α production: raising it slightly at low and reducing at higher concentration. By contrast, Tau had no significant effect on the cytokine production. These results indicate that Tau-Cl modulates synthesis of pro-inflammatory cytokines, and therefore it may play a role in the initiation and propagation of immune response. Received November 29, 2001 Accepted January 18, 2002 Published online August 30, 2002 Acknowledgments This research was supported by grants from the State Committee for Scientific Research of Poland (No 4 P05B 01018) and the Institute of Rheumatology (No I/14). The Institute of Rheumatology is supported by a core grant from the State Committee for Scientific Research of Poland. Authors' address: Ewa Kontny, Ph.D., Department of Pathophysiology and Immunology, Institute of Rheumatology, Spartanska 1, 02-637 Warsaw, Poland, E-mail: zpatiir@warman.com.pl Abbreviations: Tau, taurine; Tau-Cl, taurine chloramine; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; IL-1β, interleukin 1β; IL-6, interleukin 6; PBMC, peripheral blood mononuclear cells     

The role of Card9 overexpression in peripheral blood mononuclear cells from patients with aseptic acute pancreatitis

Activated mononuclear cells are an early event in the course of severe acute pancreatitis (SAP). To date, the molecular mechanism triggering peripheral blood mononuclear cells (PBMCs) is poorly understood. The aim of this paper was to determine the potential role of Card9 in SAP. We collected data from 72 subjects between January 2013 and June 2014. Subsequently, PBMCs were isolated on day 1, 3 and 5 of pancreatitis. Immunofluorescence staining, quantitative real‐time PCR, Western blotting, immunoprecipitation and ELISA were used to determine the role of Card9 in SAP. Microbial culture showed that SAP patients at the early period did not develop any bacteria and fungi infection. Card9 expression in SAP patients was higher than that in mild acute pancreatitis and volunteer healthy controls, up to the peak on day 1. The monocyte‐derived cytokines interleukin (IL)‐17, IL‐1β, IL‐6 and tumour necrosis factor‐α mediated by the induction of Card9 markedly increased in SAP patients compared with the control group. Furthermore, the inducible formation of Card9‐Bcl10 complex was found in PBMCs, which may be involved in nuclear factor kappa B (NF‐κB) and p38 activation in SAP. Receiver operating characteristic curve indicated that Card9 levels had a high sensitivity of 87.5% and specificity of 67.7%, showing the close correlation with SAP patients. Card9 overexpression was firstly found in aseptic SAP, which may be played an important role in NF‐κB and p38 activation in PBMCs. It also provided the new insights into therapeutic interventions by targeting monocytes activation in SAP patients.     

RNa-polymerase,type I: Activity in rat brain cell nuclei and peripheral blood mononuclear cells after Venezuelan Equine Encephalomyelitis virus infection

Rats inoculated intraperitoneally with 100 LD₅₀ of Venezuelan Equine Encephalomyelitis virus (VEE), showed a significant decrease of DNA-dependent RNA polymerase type I activity of brain nuclei of 29.7% and 59.3% at 24 and 48 hours after infection, respectively, while the animals had no clinical symptoms of illness. No alterations were observed in the nuclei of mononuclear cells at any time. VEE virus titer was higher in the serum than in the brain. The results suggest that viral infection produced a modification in the activity of this enzyme only in brain, even with a low amount of virus, and had no effect on enzyme activity of mononuclear cells.     

柯萨奇病毒 A16在恒河猴外周血单个核细胞中的增殖研究

柯萨奇病毒A16（CA16）感染灵长类动物模型显示该病毒在猴体内可形成明显的病毒血症期,在此基础上,本研究进一步分析病毒血症形成与CA16在外周血单个核细胞（PBMC ）不同组分中增殖的关系。首先用CA16感染恒河猴,检测病毒在PBMC组分CD4＋、CD8＋、CD20＋、CD11c＋、CD16＋和CD14＋细胞中的增殖情况和感染动力学,发现病毒仅在部分CD14＋细胞中有增殖表现。然后通过刺激CD14＋细胞产生一定量的树突细胞（DC）,用CA16感染DC ,发现 CA16能在DC中形成具有动力学意义的增殖过程,增殖峰值出现在感染后12～36 h。这些结果可能为CA16感染发病机制提供重要信息。     

Involvement of IL-10 in the suppression of antibody production by in vitro immunized peripheral blood mononuclear cells

Previously, we have established an in vitro immunization method to induce antigen-specific antibody-producing B cells. In the present study, we have attempted to clarify the mechanisms that regulate antibody production by in vitro immunized peripheral blood mononuclear cells (PBMC). Freshly isolated PBMC did not induce antibody production following in vitro immunization, but expressed the interleukin (IL)-10 gene. On the other hand, PBMC pretreated with l-leucyl-l-leucine methyl ester (LLME) induced antibody production, but did not express the IL-10 gene. IL-10 induced functional impairment of CD4⁺ Th cells and CD11c⁺ DC, resulting in the suppression of antibody production by in vitro immunized PBMC.