Response to Superantigen Stimulation in Peripheral Blood Mononuclear Cells from Children Perinatally Infected with Human Immunodeficiency Virus and Receiving Highly Active Antiretroviral Therapy

Our understanding of the pathogenesis of perinatal human immunodeficiency virus (HIV) infection is still evolving. We sought to characterize the response to the bacterial superantigen Staphylococcus enterotoxin B (SEB) of lymphocytes from HIV-infected children receiving treatment with highly active antiretroviral therapy (HAART). Using the flow cytometric methodology, we quantified apoptosis, proliferation, cytokine production, and activation antigen upregulation in CD4 and CD8 T lymphocytes following in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with SEB. The levels of proliferation, CD4 interleukin-2 (IL-2) production, CD8 gamma interferon (IFN-γ) production, and upregulation of CD69 expression by cells from HIV-infected children were indistinguishable from those by cells from controls. However, stimulation with SEB dramatically decreased the ratio of resting apoptotic cells to cycling apoptotic cells in the controls but not in the patients. In addition, unstimulated spontaneous apoptosis of CD4 T cells remained greater in the patients than in the controls. The percentages of IL-2-positive CD8 T cells and IFN-γ-positive CD4 T cells following SEB stimulation were significantly lower in the patients than in the controls. Our multiparameter approach was able to demonstrate differences in lymphocyte superantigen responsiveness in HIV-infected children receiving HAART in comparison to that in uninfected controls, notably, an apoptotic versus a proliferative response to stimulation.     

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.     

The Expression and Significance of TIPE2 in Peripheral Blood Mononuclear Cells from Asthmatic Children

Tumour necrosis factor‐α‐induced protein‐8 like‐2 (TIPE2) is a newly identified immune negative regulator. The abnormal expression of TIPE2 has been found in several human inflammatory diseases. However, the expression level and clinical significance of TIPE2 in childhood asthma remain unclear. In this study, we detected TIPE2 expression in peripheral blood mononuclear cells (PBMC) from 42 children with asthma and 39 healthy controls by RT‐PCR, qRT‐PCR and Western blot. We also detected the levels of serum total immunoglobulin E (IgE), eosinophil (EO), interleukin‐4 (IL‐4) and interferon‐γ (IFN‐γ) and analysed the correlations of TIPE2 expression with IgE, EO, IL‐4 and IFN‐γ. The results showed that TIPE2 mRNA and protein expression were decreased in children with asthma compared with healthy controls. The levels of IgE, EO and IL‐4 in the children with asthma were obviously higher than those in normal controls, while the level of IFN‐γ in patients with asthma was significantly lower than that in healthy subjects. Furthermore, the expression level of TIPE2 mRNA was negatively correlated with IgE, EO and IL‐4. However, no statistically significant correlation was found between TIPE2 mRNA expression and serum IFN‐γ level. In conclusion, our data suggest that reduced TIPE2 expression may contribute to the pathogenesis of childhood asthma.     

Toll-like Receptor Expression on Peripheral Blood Mononuclear Cells in Asthmatics; Implications for Asthma Management

Background

Accumulating evidence indicates that cells expressing Toll-like receptors (TLRs) play an important role in allergic diseases. The authors undertook this study to explore the hypothesis that TLR-mediated inflammatory signals are important from the perspective of asthma management.

Methods

The expressions of TLR1, TLR2, TLR3, TLR4, TLR6, and TLR9 and levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8, and IFN-γ) on the peripheral blood mononuclear cells (PBMCs) of 36 stable asthmatics on treatment (the on-treatment group), 15 asthmatics (the treatment-naïve group) before and after a 7-day course of oral prednisolone (30 mg/day), and on the PBMCs of 15 healthy controls were measured after in vitro stimulation using TLR-specific ligands.

Results

In the on-treatment group, TLR1, TLR2, TLR6, and TLR9 expressions on PBMCs were significantly different between asthmatics and controls. And the expression of TLR4 on PBMCs and TNF-α production stimulated by lipopolysaccharide (LPS), were significantly higher in mild to moderate than in severe asthmatics. Interestingly, in the treatment-naïve group, short-term prednisolone significantly increased LPS-induced TNF-α and IFN-γ productions by PBMCs.

Conclusion

TLR-mediated inflammatory signals contribute to the development and severity of asthma and are not reduced by glucocorticoid treatment, which suggests that a TLR-specific antagonist and glucocorticoid are required for the effective control of airway inflammation in asthmatics.     

新生儿和成人PBMC中IL-4基因表达和NF-AT活性的比较

通过比较新生儿脐血单个核细胞 (CBMC)和成人外周血单个核细胞 (PBMC)中IL 4基因表达水平和活化T细胞核因子(NF AT)活性 ,探讨新生儿IL 4基因表达水平低下的机制。用ELISA和RT PCR法测IL 4及其mRNA水平 ;电泳迁移率转换试验 (EMSA)测NF AT活性。结果显示 :抗CD3+PMA刺激后 ,CBMC培养上清液中IL 4水平较PBMC明显低下 (P <0 0 1) ;CBMC产生的IL 4mRNA较PBMC明显减少 ;CBMC核蛋白内几乎测不出NF AT活性 ,而PBMC核蛋白内NF AT活性较强。表明在转录水平上缺乏NF AT调控可能使新生儿IL 4基因转录减少 ,从而导致新生儿IL 4基因表达水平低下。     

Level of Human Immunodeficiency Virus DNA in Peripheral Blood Mononuclear Cells Correlates with Efficacy of Antiretroviral Therapy

A novel colorimetric assay was developed and validated for accurate quantitation of human immunodeficiency virus (HIV) DNA in peripheral blood mononuclear cells (PBMCs). We tested 318 sequential samples from 56 subjects, 53 of whom were undergoing dual or triple therapy. Patients were considered responders when viremia levels were below 5, 000 HIV RNA copies/ml. The mean DNA copy numbers for untreated and responder subjects were similar (72 and 75, respectively), while it was 4.54-fold higher for nonresponders (339). This report provides strong evidence that HIV DNA levels in PBMCs correlate with therapeutic efficacy and suggests that DNA quantitation is a useful tool to monitor the decay of the HIV reservoir toward disease remission, especially when viremia is undetectable.     

Foxp3 Expression and Nitric Oxide Production in Peripheral Blood Mononuclear Cells of Communicants with Pulmonary Tuberculosis

The understanding of the mechanisms involved in the immune response is of significant relevance to the control of tuberculosis (TB), especially in individuals living with patients with TB. To characterize the nitric oxide (NO) production and the Foxp3 marker expression in this population, peripheral blood mononuclear cells of intradomiciliary contacts of individuals with pulmonary tuberculosis with (CTb, susceptible) and without (STb, resistant) previous history of active infection were stimulated in vitro with Mycobacterium tuberculosis antigen (TbAg) and with the mitogen Concanavalin A for 24 and 48 h. The groups analysed did not present significant difference in the Foxp3 mRNA expression nor in the NO production. Negative correlation (P = 0.09) between NO and Foxp3 after a 48‐h stimulation with TbAg was observed in the STb group. In this group, after a 24‐h culture stimulated with TbAg (P = 0.03), this same correlation was observed. In comparison with the cytokines previously studied by our group (Cavalcanti et al., 2009), a positive correlation was observed between IL‐10 and Foxp3 after a 48‐h culture of cells from communicants susceptible to tuberculosis (STb) stimulated with TbAg (P = 0.04). Evaluating the entire population, a positive correlation was observed between the cytokine TNF‐α and the Foxp3 marker in the cultures stimulated for 24 (P = 0.03) and 48 (P = 0.02) hours with TbAg. Therefore, considering the similarity in the exposure and the individual capacity of responding to the contact with M. tuberculosis, the present study contributes to the comprehension of the immune regulation in individuals living with patients with TB.     

Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue

Cryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in studies of dengue. In this disease, elevated frequency of apoptotic PBMCs has been described, and molecules such as soluble tumor necrosis factor (TNF)-related apoptosis-inducing ligands (sTRAIL) are involved. This effect of dengue may affect the efficiency of PBMC cryopreservation. Here, we evaluate the viability (trypan blue dye exclusion and amine-reactive dye staining) and functionality (frequency of gamma interferon [IFN-γ]-producing T cells after polyclonal stimulation) of fresh and cryopreserved PBMCs from children with dengue (in acute and convalescence phases), children with other febrile illnesses, and healthy children as controls. Plasma sTRAIL levels were also evaluated. The frequencies of nonviable PBMCs detected by the two viability assays were positively correlated (r = 0.74; P < 0.0001). Cryopreservation particularly affected the PBMCs of children with dengue, who had a higher frequency of nonviable cells than healthy children and children with other febrile illnesses (P ≤ 0.02), and PBMC viability levels were restored in the convalescent phase. In the acute phase, an increased frequency of CD3⁺ CD8⁺ amine-positive cells was found before cryopreservation (P = 0.01). Except for B cells in the acute phase, cryopreservation usually did not affect the relative frequencies of viable PBMC subpopulations. Dengue infection reduced the frequency of IFN-γ-producing CD3⁺ cells after stimulation compared with healthy controls and convalescent-phase patients (P ≤ 0.003), and plasma sTRAIL correlated with this decreased frequency in dengue (rho = −0.56; P = 0.01). Natural dengue infection in children can affect the viability and functionality of cryopreserved PBMCs.     

Cytokine Gene Expression Occurs More Rapidly in Stimulated Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus-Infected Persons

Evaluation of cytokine gene expression following in vitro stimulation is one means of examining the dysregulation of the immune system in human immunodeficiency virus (HIV) infection. We have assessed differences in the immune status of non-HIV-infected (HIV−) and HIV-infected (HIV+) individuals by evaluating the kinetics of the expression of cytokine genes. We compared detailed time courses of cytokine mRNA expression in HIV− and HIV+ peripheral blood mononuclear cells (PBMC) and found that there is a significant shift (P < 0.01) for all cytokines examined (interleukin 2 [IL-2], IL-6, IL-10, gamma interferon, and tumor necrosis factor alpha [TNF-α]) to an earlier time of mean peak mRNA expression by HIV+ PBMC (between 4 and 8 h) compared to HIV− PBMC (8 h) in response to either phytohemagglutinin (PHA) or anti-CD3 stimulation. Additional studies showed that although PHA-stimulated HIV+ PBMC showed decreased median IL-2, IL-4, and TNF-α mRNA levels, they typically demonstrated more rapid kinetics (increased mean 4-h/24-h cytokine mRNA ratios), with significant differences for IL-4 (P < 0.05) and TNF-α (P < 0.005), compared to HIV− PBMC. The use of fresh or frozen cells gave comparable cytokine mRNA data; however, the secretion of some cytokine proteins (IL-2 receptor, IL-10, and TNF-α) appeared to be reduced in HIV+ PBMC that had been frozen and thawed. Our studies demonstrate that the kinetics of cytokine gene expression can reveal additional dysregulation of the immune system in HIV infection, suggesting that PBMC of HIV-infected persons exist in an activated state in vivo that permits them to express cytokine genes more rapidly than a normal PBMC.     

Antigen-Specific Gene Expression Profiles of Peripheral Blood Mononuclear Cells Do Not Reflect Those of T-Lymphocyte Subsets

Advances in microarray technology have allowed for the monitoring of thousands of genes simultaneously. This technology is of particular interest to immunologists studying infectious diseases, because it provides tremendous potential for investigating host-pathogen interactions at the level of immune gene expression. To date, many studies have focused either on cell lines, where the physiological relevance is questionable, or on mixed cell populations, where the contributions of individual subpopulations are unknown. In the present study, we perform an intrasubject comparison of antigen-stimulated immune gene expression profiles between a mixed population of peripheral blood mononuclear cells (PBMC) and the two predominant cell types found in PBMC, CD4⁺ and CD8⁺ T lymphocytes. We show that the microarray profiles of CD4⁺ and CD8⁺ T lymphocytes differ from each other as well as from that of the mixed cell population. The independence of the gene expression profiles of different cell types is demonstrated with a ubiquitous antigen (Candida albicans) as well as with a disease-specific antigen (human immunodeficiency virus p24). This study has important implications for microarray studies of host immunity and underscores the importance of profiling the expression of specific cell types.     

Measurement of Cytokine Secretion, Intracellular Protein Expression, and mRNA in Resting and Stimulated Peripheral Blood Mononuclear Cells

Quantitation of cytokine production is a valuable adjunct to standard immunologic assays in defining several pathologic processes. Nevertheless, there is little agreement about which tissues should be assayed, which type of assay should be performed, and which stimulation protocol should be used. As these types of assays enter the clinical arena, there is need for standardization. There is also a need to maximize the amount of information which may be derived from a single sample. We compared secreted interleukin 4 (IL-4), IL-2, IL-6, tumor necrosis factor alpha (TNF-α), and gamma interferon proteins as measured by enzyme-linked immunosorbent assay with intracellular cytokine production (IL-2 and gamma interferon) as detected by flow cytometry and quantitative competitive PCR for IL-2, IL-4, TNF-α, and gamma interferon mRNA and cDNA. Results from unstimulated cells and cells stimulated with phorbol myristate acetate, phytohemagglutinin, and phorbol myristate acetate plus phytohemagglutin were compared. All three methodologies detected significant stimulation of cytokine production. The combination of phytohemagglutinin and phorbol myristate acetate was overall the most-potent stimulus.     

Association of IL-10-Regulating MicroRNAs in Peripheral Blood Mononuclear Cells with the Pathogenesis of Autoimmune Thyroid Disease

Interleukin (IL)-10 is known to suppress inflammation in autoimmune diseases. IL-10 can be regulated by miRNAs. To elucidate the involvement of miRNAs that regulate IL-10 expression with the pathogenesis of autoimmune thyroid disease (AITD), we examined the expression levels of hsa-miR-27a-3p, hsa-miR-98-5p, hsa-miR-106a-5p, and hsa-miR-223-3p in peripheral blood mononuclear cells (PBMCs) from 43 patients with Graves’ disease (GD), 38 patients with Hashimoto’s disease (HD), and 21 healthy volunteers. We evaluated the association between the expression levels of four miRNAs and intracellular expression of IL-10 in PBMCs from 11 healthy volunteers. We also genotyped MIR27A rs895819 G/A and MIR106A rs3747440 C/G polymorphisms, which may be related to the expression of these miRNAs in 141 patients with GD, 178 patients with HD, and 84 healthy volunteers. The expression level of hsa-miR-106a-5p was significantly higher in patients with intractable GD than in those with GD in remission (p = 0.0113). The expression level of hsa-miR-223-3p was significantly lower in GD than in HD and lower in patients with intractable GD than in healthy volunteers (p = 0.0094, 0.0340). We found a negative correlation between the expression levels of hsa-miR-98-5p and the proportions of IL-10⁺ cells in stimulated PBMCs from healthy volunteers (p = 0.0092). The G allele of the MIR27A polymorphism was significantly more frequent in patients with mild HD than in healthy volunteers (p = 0.0432). In conclusion, the expression levels of hsa-miR-106a-5p and hsa-miR-223-3p were associated with the pathogenesis of AITDs. hsa-miR-98-5p may negatively regulate the expression of IL-10. The functional polymorphism of MIR27A was associated with HD severity.     

Role of Peripheral Blood Mononuclear Cells in the Predisposition of Obese Individuals to Inflammation and Infection

ObjectiveTo compare the production of pro- and anti-inflammatory cytokines by peripheral blood mononuclear cells (PBMC) from obese but otherwise healthy individuals to that of normal-weight volunteers.Methods25 healthy normal-weight subjects and 41 obese individuals were enrolled. Weight and height were measured twice. PBMC were examined for their capacity to generate pro-inflammatory (TNF-α, IFN-γ, IL-1β, IL-6, IL-2) and anti-inflammatory IL-10 and IL-1ra) cytokines.ResultsPBMC from obese individuals, compared to those from subjects with normal weight showed an increased production of the pro-inflammatory cytokines IL-2 (6.7 ± 0.4. vs. 4.9 ± 0.3 ng/ml; p = 0.003), TNF-α (505 ± 45 vs. 277 ± 32 pg/ml; p = 0.001), and IFN-γ (93.8 ± 6.0 vs. 73.9 ± 2.7 ng/ml; p = 0.0016). However, PBMC from obese individuals produced a lower amount of the anti-inflammatory cytokine IL-10 (651 ±72 pg/ml) versus those from subjects with normal weight (951 ± 133 pg/ml; p = 0.039).ConclusionsThe findings imply that obese individuals are in a ‘low-grade inflammatory state’, presumed to be connected with metabolic and cardiovascular co morbidities. The surplus of pro-inflammatory cytokines produced by circulating mononuclear cells of obese individuals, together with those secreted by adipocytes and non-fat cells in the adipose tissue, may contribute to the predisposition of obese patients to inflammation and infections.Key Words: Obesity, Peripheral blood mononuclear cells, Cytokines, Inflammation, Infection     

系统性红斑狼疮外周血单个核细胞cFLIP-L mRNA和cFLIP-S mRNA表达的研究

目的 探讨系统性红斑狼疮(system lupus erythematosus,SLE)患者外周血单个核细胞(peripheral blood monouuclear cells,PBMC)中细胞型Fas相关死亡域样白介素-1β转换酶抑制蛋白(cFLIP)表达的意义.方法 应用半定量RT-PCR方法检测38例SLE患者和21名正常人PBMC中cFLIP-L mRNA和cFLIP-S mRNA的表达水平,并与SLE疾病活动指数(SLEDAI)评分进行相关性分析.结果 ① SLE患者PBMC中cFLIP-L mRNA和cFLIP-S mRNA表达水平均明显高于正常对照组(P＜0.01);SLE患者活动组cFLIP-L mRNA表达水平显著高于非活动组(P＜0.05),cFLIP-S mRNA表达水平在SLE患者活动组与非活动组之间没有显著性差异(P＞0.05). ② SLE患者cFLIP-L mRNA表达水平与SLEDAI评分呈正相关(r=0.423,P＜0.01);而cFLIP-S mRNA表达水平与SLEDAI评分无明显相关性(r=0.270,P＞0.05).结论 cFLIP-L mRNA和cFLIP-S mRNA可能在SLE发病机制中起重要作用.