Response to Rhinovirus Infection by Human Airway Epithelial Cells and Peripheral Blood Mononuclear Cells in an In Vitro Two-Chamber Tissue Culture System

Human rhinovirus (HRV) infections are associated with the common cold, occasionally with more serious lower respiratory tract illnesses, and frequently with asthma exacerbations. The clinical features of HRV infection and its association with asthma exacerbation suggest that some HRV disease results from virus-induced host immune responses to infection. To study the HRV-infection-induced host responses and the contribution of these responses to disease, we have developed an in vitro model of HRV infection of human airway epithelial cells (Calu-3 cells) and subsequent exposure of human peripheral blood mononuclear cells (PBMCs) to these infected cells in a two-chamber trans-well tissue culture system. Using this model, we studied HRV 14 (species B) and HRV 16 (species A) induced cytokine and chemokine responses with PBMCs from four healthy adults. Infection of Calu-3 cells with either virus induced HRV-associated increases in FGF-Basic, IL-15, IL-6, IL-28A, ENA-78 and IP-10. The addition of PBMCs to HRV 14-infected cells gave significant increases in MIP-1β, IL-28A, MCP-2, and IFN-α as compared with mock-infected cells. Interestingly, ENA-78 levels were reduced in HRV 14 infected cells that were exposed to PBMCs. Addition of PBMCs to HRV 16-infected cells did not induce MIP-1β, IL-28A and IFN-α efficiently nor did it decrease ENA-78 levels. Our results demonstrate a clear difference between HRV 14 and HRV 16 and the source of PBMCs, in up or down regulation of several cytokines including those that are linked to airway inflammation. Such differences might be one of the reasons for variation in disease associated with different HRV species including variation in their link to asthma exacerbations as suggested by other studies. Further study of immune responses associated with different HRVs and PBMCs from different patient groups, and the mechanisms leading to these differences, should help characterize pathogenesis of HRV disease and generate novel approaches to its treatment.     

Increased Responsiveness of Peripheral Blood Mononuclear Cells to In Vitro TLR 2, 4 and 7 Ligand Stimulation in Chronic Pain Patients

Glial activation via Toll-like receptor (TLR) signaling has been shown in animals to play an important role in the initiation and establishment of chronic pain. However, our ability to assess this central immune reactivity in clinical pain populations is currently lacking. Peripheral blood mononuclear cells (PBMCs) are an accessible source of TLR expressing cells that may mirror similarities in TLR responsiveness of the central nervous system. The aim of this study was to characterize the IL-1β response to various TLR agonists in isolated PBMCs from chronic pain sufferers (on and not on opioids) and pain-free controls. Venous blood was collected from 11 chronic pain sufferers on opioids (≥ 20 mg of morphine / day), 8 chronic pain sufferers not on opioids and 11 pain-free controls. PBMCs were isolated and stimulated in vitro with a TLR2 (Pam3CSK4), TLR4 (LPS) or TLR7 (imiquimod) agonist. IL-1β released into the supernatant was measured with ELISA. Significantly increased IL-1β expression was found in PBMCs from chronic pain sufferers (on and not on opioids) compared with pain-free controls for TLR2 (F _{(6, 277)} = 15, P<0.0001), TLR4 (F _{(8, 263)} = 3, P = 0.002) and TLR7 (F _(2,201) = 5, P = 0.005) agonists. These data demonstrate that PBMCs from chronic pain sufferers were more responsive to TLR agonists compared with controls, suggesting peripheral cells may have the potential to become a source of biomarkers for chronic pain.     

成人外周血来源树突状细胞体外诱导培养动态监测

目的:分析体外诱导培养树突状细胞(dendritic cells,DCs)表面分子及成熟度的动态变化;方法:利用流式细胞仪(fluorescence activated cell sorter,FACS)检测分析DCs表面成熟标志分子CD83和T细胞辅助分子CD58、CD54、CD40、CD80、CD86、HLA-DR、HLA-ABC表达水平;结果:体外诱导培养5 d细胞即高表达DCs标志分子CD11c,细胞成熟度与细胞表面T细胞辅助分子的表达水平随培养天数的增加有一定提高,但显著低于大肠杆菌LPS刺激的DCs;结论:诱导培养6 d DCs表型为CD83 low、CD58low、CD54 low、CD40 low、CD80 low、CD86 low、HLA-DRhigh、HLA-ABChigh,为不成熟DCs.     

伏马菌素B1对人外周血单个核细胞抗原加工相关转运子表达的影响

探讨伏马菌素B1(FB1)对体外培养的人外周血单个核细胞(hPBMC)抗原加工相关转运子(TAP-1)表达的影响。采用流式细胞定量检测(FCM)、免疫印迹(Western印迹)及半定量RT-PCR方法,研究不同浓度FB1(0,10和50μmol/L)处理后人外周血单个核细胞TAP-1在mRNA和蛋白质水平表达的影响。RT-PCR检测结果表明,10和50μmol/L FB1处理24h后,处理组细胞TAP-1mRNA明显低于对照组。在蛋白质水平,FCM定量分析表明,两个处理组细胞表面TAP1的平均荧光强度均较对照组降低,以50μmol/LFB1处理组降低显著(P<0.05)。免疫印迹结果亦表明,FB1处理组TAP-1的表达均较对照组降低。10和50μmol/LFB1可抑制体外培养的hPBMCTAP-1mRNA和蛋白质表达。     

脱氧雪腐镰刀菌烯醇抑制体外培养人外周血单个核细胞低分子量蛋白酶体-2表达

探讨脱氧雪腐镰刀菌烯醇(DON)对人外周血单个核细胞参与抗原呈递的低分子量蛋白酶体-2(LMP-2)表达的影响。采用流式细胞术(FCM)和半定量RT-PCR方法从蛋白质和mRNA水平分析了不同剂量DON对体外培养人外周血单个核细胞LMP-2分子表达的影响及其量效关系。FCM定量检测结果表明,不同浓度DON处理均可一定程度抑制人外周血单个核细胞LMP-2的表达,50ng/mlDON组、100ng/mlDON组、1000ng/mlDON组和2000ng/mlDON组LMP-2平均荧光强度分别为6.99±0.72、6.21±0.55、5.34±0.56和5.03±0.43,在50～2000ng/mL范围内随着DON浓度增加,外周血单个核细胞LMP-2表达降低,与DON浓度呈显著负相关(r=0.824,P<0.01)。半定量RT-PCR结果显示,不同浓度DON处理均可抑制人外周血单个核细胞LMP-2mRNA表达。DON在蛋白质和mRNA水平可剂量依赖地抑制体外培养的人外周血单个核细胞LMP-2的表达。     

Cladribine Exposure Results in a Sustained Modulation of the Cytokine Response in Human Peripheral Blood Mononuclear Cells

Background and Objectives

Cladribine is a cytotoxic drug which ameliorates the clinical course of relapsing-remitting multiple sclerosis. In addition to cytotoxicity, the mode of action may include immunomodulatory mechanisms. This in vitro study was designed to investigate cladribine’s effects on cell function after the removal of cladribine to distinguish cytotoxic versus immunomodulatory effects.

Methods

Cells were incubated in the absence or presence of cladribine (1×10^-8 M to 1×10^-5 M) for 72 h. Cladribine was removed from the cell culture and surviving peripheral blood mononuclear cells were cultured up to 58 days to determine the immunomodulatory effects of cladribine on cell function (e.g., proliferation and cytokine release).

Results

In the long-term, brief cladribine exposure did not impair the proliferation of surviving peripheral blood mononuclear cells. However, it induced an anti-inflammatory shift in the cytokine milieu with significantly enhanced release of IL-4 (Days 9 and 44, p<0.01; Day 58, p<0.05) and IL-5 (Day 9, p<0.01), resulting in an increased IL-4/INF-gamma ratio (Days 9 and 44, p<0.01; Day 58, p<0.05). Additionally, a trend towards an increased IL-10 production was observed. No changes were found in the production of IFN-gamma, TNF-alpha, IL-6, IL-8, IL-17A, IL-23 or NGF-beta.

Conclusions

In vitro cladribine exposure induces a sustained anti-inflammatory shift in the cytokine profile of surviving peripheral blood mononuclear cells. This immunomodulatory action might contribute to cladribine’s beneficial effects in the treatment of multiple sclerosis.     

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.     

Merkel Cells as Putative Regulatory Cells in Skin Disorders: An In Vitro Study

Merkel cells (MCs) are involved in mechanoreception, but several lines of evidence suggest that they may also participate in skin disorders through the release of neuropeptides and hormones. In addition, MC hyperplasias have been reported in inflammatory skin diseases. However, neither proliferation nor reactions to the epidermal environment have been demonstrated. We established a culture model enriched in swine MCs to analyze their proliferative capability and to discover MC survival factors and modulators of MC neuroendocrine properties. In culture, MCs reacted to bFGF by extending outgrowths. Conversely, neurotrophins failed to induce cell spreading, suggesting that they do not act as a growth factor for MCs. For the first time, we provide evidence of proliferation in culture through Ki-67 immunoreactivity. We also found that MCs reacted to histamine or activation of the proton gated/osmoreceptor TRPV4 by releasing vasoactive intestinal peptide (VIP). Since VIP is involved in many pathophysiological processes, its release suggests a putative regulatory role for MCs in skin disorders. Moreover, in contrast to mechanotransduction, neuropeptide exocytosis was Ca²⁺-independent, as inhibition of Ca²⁺ channels or culture in the absence of Ca²⁺ failed to decrease the amount of VIP released. We conclude that neuropeptide release and neurotransmitter exocytosis may be two distinct pathways that are differentially regulated.     

Epstein-Barr Virus Encoded dUTPase Containing Exosomes Modulate Innate and Adaptive Immune Responses in Human Dendritic Cells and Peripheral Blood Mononuclear Cells

We have recently demonstrated that Epstein-Barr virus (EBV)-encoded deoxyuridine triphosphate nucleotidohydrolase (dUTPase) modulates innate immunity in human primary monocyte-derived macrophages through toll-like receptor (TLR) 2 leading to NF-κB activation and the production of pro-inflammatory cytokines. Our previous depletion studies indicated that dendritic cells (DCs) may also be a target of the EBV-encoded dUTPase. However, the role of EBV-encoded dUTPase in DC activation/function and its potential contribution to the inflammatory cellular milieu characteristic of EBV-associated diseases remains poorly understood. In the present study, we demonstrate that EBV-encoded dUTPase significantly altered the expression of genes involved in oncogenesis, inflammation and viral defense mechanisms in human primary DCs by microarray analysis. Proteome array studies revealed that EBV-encoded dUTPase modulates DC immune responses by inducing the secretion of pro-inflammatory T_H1/T_H17 cytokines. More importantly, we demonstrate that EBV-encoded dUTPase is secreted in exosomes from chemically induced Raji cells at sufficient levels to induce NF-κB activation and cytokine secretion in primary DCs and peripheral blood mononuclear cells (PBMCs). Interestingly, the production of pro-inflammatory cytokines in DCs and PBMCs was TLR2-dependent. Together these findings suggest that the EBV-encoded dUTPase may act as an intercellular signaling molecule capable of modulating the cellular microenvironment and thus, it may be important in the pathophysiology of EBV related diseases.     

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.     

An Air-Drying Technique for Flattening Chromosomes in Mammalian Cells Grown In Vitro

The method described was developed to facilitate the analysis of chromosome complements in cells freshly isolated from monkey kidney cortex and grown on glass, and in “altered” monkey cells grown on glass or in suspension. Cells were treated with hypotonic solution (quarter-strength Tyrode or diluted medium) for 30 min, or with colchicine in a final concentration of 25 μg/ml (.0025%) for 12-18 hr followed by hypotonic salt solution for 5 min, then fixed in acetic alcohol (1:3) for 5 min. With cells centrifuged from suspended cultures, addition of fixative had to be gradual. Directly after fixation, films of cells on slides were air dried completely. This produces a more uniform and complete flattening of cells than can be achieved by manual pressure; yet, fragmentation of chromosome complements does not occur. Fixed and air dried slides may be stored for days without deterioration or they may be stained immediately in 2% natural orcein (G. T. Gurr, London) in 50% acetic acid. Preparations can be made permanent by a dry ice schedule, without loss, shrinkage, or distortion of cells.     

Effects of Specific Gangliosides on the In Vitro Proliferation of MPTP-Susceptible Cells

Abstract: To determine which portion of a ganglioside molecule might be necessary for the enhancement of recovery from MPTP-induced lesions, the ability of specific gangliosides to stimulate proliferation of MPTP-treated 140–3 cells was investigated. The results indicate that of the gangliosides tested, GM1 was the most effective. Although GD1 a and GT1 b were able to enhance the proliferation of MPTP-treated cells, twice as much GT1b was needed to induce the same effect seen with GM1. In contrast, asialo-GM1, GM2, and GM3 were ineffective at promoting proliferation of MPTP-treated cells. The isolated oligosaccharide of GM1 had little effect. These results indicate that in addition to the sialosyl residue, at least the Gal(β1–3)Gal-NAc portion of the oligosaccharide chain and the ceramide moiety are essential for the induction of proliferation of the MPTP-treated cells. Investigation of the time of addition of GM1 on its ability to counteract the MPTP-induced inhibition of 140–3 cell proliferation indicated that addition of GM1 before or concomitantly with MPTP resulted in a significant reduction in MPTP-induced inhibition of cell proliferation.     

Proliferative Response of Human Peripheral Blood Mononuclear Cells to Japanese Encephalitis Virus

Cell-mediated immune response to Japanese encephalitis virus (JEV) and its purified envelope (E) protein was measured in 45 laboratory confirmed JE patients using a proliferation assay of peripheral blood mononuclear cells (PBMC). In parallel, JEV-specific IgM antibodies were measured by ELISA. No correlation was observed between the antibody response and results of the lymphocyte proliferation assay. Only 11 of the 42 patients positive in the antibody test were positive in the proliferation assay, and PBMC from 14/45 (31%) patients did not respond to either phytohemagglutinin and to JEV and/or its purified E protein antigen. No correlation was observed between the cell-mediated immune response and the final clinical outcome (fatality vs. recovery).     

Impact of Spermine on Down-Regulation of Th2 Cytokines in Peripheral Blood Mononuclear Cells from Cedar Pollinosis Subjects in Vitro

The effects of spermine (SPM) on the production of Th1 and Th2 cytokines using peripheral blood mononuclear cells (PBMCs) derived from cedar pollinosis subjects were investigated. Th2 cytokines produced by PBMCs stimulated by 10 μM SPM was significantly lower than that in the controls. These findings indicate that blood SPM is an important factor in the Th1/Th2 balance.     

Reprogrammed Peripheral Blood Mononuclear Cells are Able to Survive Longer in Irradiated Female Mice

Induced multipotent stem (iMS) cells are originated from somatic cells and become multipotent by genetic and/or epigenetic modifications. Previous studies have shown that the fish oocytes extracts (FOE) can induce skin fibroblast cells into iMS cells. In this study, we aim to determine whether FOE can similarly induce mouse peripheral blood mononuclear cells (PBMCs) into the iMS state and if so, whether they can survive longer when they are transplanted into the irradiation female mice. PBMCs of GFP-transgenic male mice were cultured and transiently reprogrammed by FOE. They were deemed reaching the iMS state after detection of expression of stem cell markers. The iMS-like PBMCs were transplanted into female C57BL mice by tail vein injection. The spleen wet weights as well as numbers of colonies of the recipient mice were examined. The results showed the spleen wet weights and numbers of spleen colonies of FOE-induced group were all significantly higher than those of the non-induced group and negative control group. On day 90 after transplantation, FISH analysis detected the presence of Y chromosome in the induced group, but not of the other groups. The current findings demonstrate that FOE-induced PBMCs are able to survive longer in irradiated female mice.