mRNA Expression In Peripheral Blood Mononuclear Cells Research Articles

Cytokine mRNA profiling and the proliferative response of bovine peripheral blood mononuclear cells to Mycoplasma bovis

Mycoplasma bovis is known as a significant pathogen and cause of large economic losses in beef and dairy calves worldwide. Numerous factors appear to play an important role in the development of disease during infection with M. bovis, e.g., inhibition of immune cell proliferation and induction of lymphocyte apoptosis. However, the mechanisms involved in M. bovis infections have not been explored and remain incompletely understood. We investigated the major cytokine mRNA expression in bovine PBMC stimulated with M. bovis, for comparison, Staphylococcus aureus and Escherichia coli, which are the representative mastitis-causing pathogens. Here we demonstrated that live M. bovis significantly induced tumor necrosis factor alpha (TNF-α), interleukin 12p40 (IL-12), and interferon gamma (IFN-γ) mRNA expression in bovine peripheral blood mononuclear cells (PBMC) at a multiplicity of infection (MOI) of 1000 but not at an MOI of 10 and 100. Live M. bovis at MOIs of 1, 10, and 100 induced significant bovine PBMC proliferative responses compared with unstimulated bovine PBMC. Furthermore, we showed that the cultural supernatant of M. bovis induced a significant increase in TNF-α, IL-6, and IL-10 mRNA expression in bovine PBMC. Our results suggest that M. bovis weakly affects the cellular integrity of bovine PBMC and induces clear proliferative responses and associated cytokine production in them. However, large numbers of live M. bovis are required to induce an immune response in bovine PBMC.

Association Between Copy Number Variations of TLR7 and Ocular Behçet's Disease in a Chinese Han Population.

The purpose of this study was to test whether gene copy number variations (CNVs) of Toll-like receptors (TLRs) are associated with uveitis. Copy number variations of TLRs were detected by real-time PCR. The first stage of the study consisted of enrolling 400 Behçet's disease (BD) patients, 400 Vogt-Koyanagi-Harada syndrome patients, 400 patients with acute anterior uveitis associated with or without ankylosing spondylitis, and 600 healthy subjects. The second stage included another set of 578 BD patients and 1000 healthy controls. The frequencies of TLR gene copy number types (TLR1, TLR2, TLR3, TLR5, TLR6, TLR7, TLR9, TLR10) were compared among patients and controls by using the χ(2) test. Real-time PCR was used to detect mRNA expression from peripheral blood mononuclear cells (PBMCs) obtained from healthy controls following stimulation with the TLR7 agonist R848. Levels of TNF-α, IL-6, IL-1β, and IFN-β in culture supernatants were measured by ELISA. All TLRs tested, except for TLR7, had a gene copy number of two in more than 98% of individuals tested. In the first stage, we found a significantly increased frequency of more than one copy of TLR7 (located on the X chromosome) in male BD patients and more than two copies in female patients (correction of P value [PC] = 0.021; PC = 0.048, respectively). A second stage and combined study confirmed the association (PC = 1.14 × 10(-6); PC = 9.12 × 10(-5), respectively). TLR7 mRNA expression in PBMCs was increased in healthy male carriers having more than one copy of TLR7 or females having more than two copies following stimulation with R848 (P = 0.021, P = 0.006, respectively). No effect of the various TLR7 copies on the release of TNF-α, IL-6, IL-1β, and IFN-β could be detected. This study provides evidence that a high copy number of TLR7 confers risk for BD in a Chinese Han population. (http://www.chictr.org number, ChiCTR-CCC-12002184.).

Increased IL-21/IL-21R expression and its proinflammatory effects in autoimmune thyroid disease

To determine the potential role of interleukin-21 (IL-21) / IL-21 receptor (IL-21R) in the pathogenesis of autoimmune thyroid disease (AITD) mainly known as Graves’ disease (GD) and Hashimoto’s thyroiditis (HT). IL-21 and IL-21R of peripheral blood samples and/or thyroid tissues from AITD patients and healthy controls were analyzed by ELISA, quantitative real-time polymerase chain reaction (qRT-PCR), flow cytometry and immunohistochemistry. In vitro, the mRNA and protein of inflammatory cytokines of cultured peripheral blood mononuclear cells (PBMCs) upon recombinant human IL-21 (rhIL-21) stimulation were detected. There was an increased serum concentration of IL-21 in untreated GD and HT patients, and IL-21+CD3+CD8−T cells were significantly increased in PBMCs of HT patients compared with healthy volunteers. The IL-21 mRNA expression in PBMCs increased dramatically in GD and HT patients, and marked augmentations of IL-21 and IL-21R mRNA in thyroid tissues of HT patients were observed. Immunohistochemical staining confirmed the expression of IL-21R protein in HT thyroid cells and lymphocytes. In vitro, PBMCs from GD cultured with rhIL-21 induced increased IL-17A but decreased IL-4 production, while from HT stimulated by rhIL-21 induced augmented production of IFN-γ. In conclusion, the expression of IL-21 and IL-21R were up-regulated in AITD and may be involved in the pathogenesis of the disease through augmenting aberrant immune cascade.

Peripheral Blood Mononuclear Cell Gene Expression Remains Broadly Altered Years after Successful Interferon-Based Hepatitis C Virus Treatment

Background. Inflammatory gene expression in peripheral blood mononuclear cells (PBMCs) is altered in chronic Hepatitis C Virus (HCV) infection. Duration of changes after pegylated interferon- (peg-IFN-) based HCV treatment is unclear. Methods. PBMC mRNA expression of 184 inflammatory response genes was analyzed (nCounter GX Human Inflammation Kit, Nanostring) from peg-IFN treatment nonresponders (NR, n = 18), sustained virologic responders (SVR, n = 22), and spontaneous clearers (SC, n = 15). Logistic regression was used for comparison. Results. Median time from last treatment was 2 and 2.7 years in SVR and NR, respectively (p = NS). Mean mRNA counts were significantly different for 42 and 29 genes comparing SVR to SC patients and NR to SC, respectively, and no genes comparing SVR to NR. Differential expression of 24 genes was significantly different in both SVR and NR groups compared to SC. Among these 24 acute and chronic inflammatory cascade genes, significant upregulation was noted for proinflammatory transcription regulators Fos, CEBPB, and MyD88 in SVR and NR compared to SC. HDAC4 was significantly downregulated in SVR and NR compared to the SC group. Conclusions. PBMC inflammatory gene expression patterns in SVR resemble NR more than SC patients. A generalized inflammatory response persists in PBMCs long after successful peg-IFN treatment for HCV infection.

P064 : KIR mRNA EXPRESSION IN NATURAL KILLER CELLS AND PERIPHERAL BLOOD MONONUCLEAR CELLS ASSAYED BY QUANTITATIVE REAL-TIME PCR

Aim To develop assays to quantify the mRNA transcribed from 15 KIR functional genes utilizing a quantitative real-time PCR (qPCR) method. To study the KIR mRNA expression patterns in NK cells and peripheral blood mononuclear cells (PBMC) among 50 healthy volunteers. To correlate the qPCR results from NK cells and PBMC. Methods PBMC were obtained from freshly drawn whole blood (maximum 48 h) from 50 healthy volunteers using a Ficoll method. NK cell enrichment from PBMC was performed by negative selection using a magnetic bead kit. mRNA extraction and cDNA synthesis was followed by qPCR using EvaGreen dye. Relative quantification (RQ) of KIR mRNA expression was quantified by 2-ΔΔCt algorithm with GAPDH as the housekeeping gene and a commercial human cDNA as the universal reference. Results 16 mRNA expression assays for 15 KIR genes, including assays for both the full-length and truncated variants for 2DS4, have been developed and validated. Among the 50 individuals, KIR mRNAs were detected in all samples carrying respective KIR genes and were negative for the KIR gene negative samples tested. In particular, 2DL1, 2DL4 and 3DL2 were positive for all 50 individuals at both genomic DNA and mRNA levels, whereas mRNA was undetected for all 2DL5B and 3DL3 gene positive samples. Among 50 individuals, the most variably expressed KIR mRNA was 2DL1 in NK cells and 3DL1 in PBMC. The strongest correlation between NK cells and PBMC was found for 2DS5 ( R = 0.9582, n = 9, p Conclusions We established qPCR assays to quantify the mRNA expression of 15 KIR genes. KIR mRNA expression varied among healthy individuals carrying the same KIR genotype. Analyzing NK cells and PBMC will allow establishing NK cell specific and overall KIR mRNA expression signatures from an individual. Results from NK cells and PBMC are correlated to a different degree for the 15 KIR genes, confirming that NK cells are the main predictor for KIR mRNA expression in PBMC. However, any KIR mRNA expression in PBMC is only partially reflective of the KIR mRNA expression in NK cells.

Infection with host-range mutant adenovirus 5 suppresses innate immunity and induces systemic CD4+ T cell activation in rhesus macaques.

Ad5 is a common cause of respiratory disease and an occasional cause of gastroenteritis and conjunctivitis, and seroconversion before adolescence is common in humans. To gain some insight into how Ad5 infection affects the immune system of rhesus macaques (RM) 18 RM were infected with a host-range mutant Ad5 (Ad5hr) by 3 mucosal inoculations. There was a delay of 2 to 6 weeks after the first inoculation before plasmacytoid dendritic cell (pDC) frequency and function increased in peripheral blood. Primary Ad5hr infection suppressed IFN-γ mRNA expression, but the second Ad5hr exposure induced a rapid increase in IFN-gamma mRNA in peripheral blood mononuclear cells (PBMC). Primary Ad5hr infection suppressed CCL20, TNF and IL-1 mRNA expression in PBMC, and subsequent virus exposures further dampened expression of these pro-inflammatory cytokines. Primary, but not secondary, Ad5hr inoculation increased the frequency of CXCR3+ CD4+ T cells in blood, while secondary, but not primary, Ad5hr infection transiently increased the frequencies of Ki67+, HLADR+ and CD95+/CCR5+ CD4+ T cells in blood. Ad5hr infection induced polyfunctional CD4 and CD8+ T cells specific for the Ad5 hexon protein in all of the animals. Thus, infection with Ad5hr induced a complex pattern of innate and adaptive immunity in RM that included transient systemic CD4+ T cell activation and suppressed innate immunity on re-exposure to the virus. The complex effects of adenovirus infection on the immune system may help to explain the unexpected results of testing Ad5 vector expressing HIV antigens in Ad5 seropositive people.

The mitochondrial cardiolipin remodeling enzyme lysocardiolipin acyltransferase is a novel target in pulmonary fibrosis.

Lysocardiolipin acyltransferase (LYCAT), a cardiolipin-remodeling enzyme regulating the 18:2 linoleic acid pattern of mammalian mitochondrial cardiolipin, is necessary for maintaining normal mitochondrial function and vascular development. We hypothesized that modulation of LYCAT expression in lung epithelium regulates development of pulmonary fibrosis. To define a role for LYCAT in human and murine models of pulmonary fibrosis. We analyzed the correlation of LYCAT expression in peripheral blood mononuclear cells (PBMCs) with the outcomes of pulmonary functions and overall survival, and used the murine models to establish the role of LYCAT in fibrogenesis. We studied the LYCAT action on cardiolipin remodeling, mitochondrial reactive oxygen species generation, and apoptosis of alveolar epithelial cells under bleomycin challenge. LYCAT expression was significantly altered in PBMCs and lung tissues from patients with idiopathic pulmonary fibrosis (IPF), which was confirmed in two preclinical murine models of IPF, bleomycin- and radiation-induced pulmonary fibrosis. LYCAT mRNA expression in PBMCs directly and significantly correlated with carbon monoxide diffusion capacity, pulmonary function outcomes, and overall survival. In both bleomycin- and radiation-induced pulmonary fibrosis murine models, hLYCAT overexpression reduced several indices of lung fibrosis, whereas down-regulation of native LYCAT expression by siRNA accentuated fibrogenesis. In vitro studies demonstrated that LYCAT modulated bleomycin-induced cardiolipin remodeling, mitochondrial membrane potential, reactive oxygen species generation, and apoptosis of alveolar epithelial cells, potential mechanisms of LYCAT-mediated lung protection. This study is the first to identify modulation of LYCAT expression in fibrotic lungs and offers a novel therapeutic approach for ameliorating lung inflammation and pulmonary fibrosis.

Elevated HERV-K mRNA expression in PBMC is associated with a prostate cancer diagnosis particularly in older men and smokers.

Aberrant expression of subgroup k human endogenous retroviruses (HERV-K) has been observed in prostate cancer. This subgroup is unique because it encodes sequences in the human genome containing open reading frames for near intact retroviruses. We hypothesized that HERV-K reactivation could serve as a non-invasive early disease detection marker for prostate cancer. We evaluated HERV-K gag messenger RNA (mRNA) expression in blood samples of African-American and European-American men using a case-control design via quantitative real-time PCR. Additionally, we examined HERV-K envelope protein expression in prostate tumors by immunohistochemistry. HERV-K envelope protein was commonly upregulated in prostate tumors, but more so in tumors of African-American than European-American patients (61% versus 40%, P < 0.01). Examining HERV-K gag expression in peripheral blood mononuclear cells (PBMC) from 294 cases and 135 healthy men, we found that the abundance of HERV-K gag message was significantly higher in cases than controls and was associated with increased plasma interferon-γ. Men with gag expression in the highest quartile had >12-fold increased odds {odds ratio = 12.87 [95% confidence interval 6.3-26.25]} of being diagnosed with prostate cancer than those in the lowest quartile. Moreover, our results showed that HERV-K expression may perform better as a disease biomarker in older than younger men (whereas the sensitivity of prostate-specific antigen (PSA) testing decreases with age) and in men with a smoking history compared with never smokers. Combining non-invasive HERV-K testing with PSA testing may improve the efficacy of prostate cancer detection specifically among older men and smokers who tend to develop a more aggressive disease.

Interleukin 32γ (IL-32γ) is highly expressed in cutaneous and mucosal lesions of American Tegumentary Leishmaniasis patients: association with tumor necrosis factor (TNF) and IL-10.

BackgroundThe interleukin 32 (IL-32) is a proinflammatory cytokine produced by immune and non-immune cells. It can be induced during bacterial and viral infections, but its production was never investigated in protozoan infections. American Tegumentary Leishmaniasis (ATL) is caused by Leishmania protozoan leading to cutaneous, nasal or oral lesions. The aim of this study was to evaluate the expression of IL-32 in cutaneous and mucosal lesions as well as in peripheral blood mononuclear cells (PBMC) exposed to Leishmania (Viannia) braziliensis.MethodsIL-32, tumour necrosis factor (TNF) and IL-10 protein expression was evaluated by immunohistochemistry in cutaneous, mucosal lesions and compared to healthy specimens. The isoforms of IL-32α, β, δ, γ mRNA, TNF mRNA and IL-10 mRNA were assessed by qPCR in tissue biopsies of lesions and healthy skin and mucosa. In addition, PBMC from healthy donors were cultured with amastigotes of L. (V.) braziliensis. In lesions, the parasite subgenus was identified by PCR-RFLP.ResultsWe showed that the mRNA expression of IL-32, in particular IL-32γ was similarly up-regulated in lesions of cutaneous (CL) or mucosal (ML) leishmaniasis patients. IL-32 protein was produced by epithelial, endothelial, mononuclear cells and giant cells. The IL-32 protein expression was associated with TNF in ML but not in CL. IL-32 was not associated with IL-10 in both CL and ML. Expression of TNF mRNA was higher in ML than in CL lesions, however levels of IL-10 mRNA were similar in both clinical forms. In all lesions in which the parasite was detected, L. (Viannia) subgenus was identified. Interestingly, L. (V.) braziliensis induced only IL-32γ mRNA expression in PBMC from healthy individuals.ConclusionsThese data suggest that IL-32 plays a major role in the inflammatory process caused by L. (Viannia) sp or that IL-32 is crucial for controlling the L. (Viannia) sp infection.

Preliminary Study on the Expression and the Clinical Significance of CD133 in Peripheral Blood of Patients with Gastric Adenocarcinoma

Background. Significances of CD133 mRNA in peripheral blood mononuclear cells (PBMCs) of gastric adenocarcinoma (GC) patients were investigated. Methods. Correlations of CD133 mRNA expression in PBMCs on clinicopathological parameters or CD133 protein expression were analyzed. Receiver operating characteristic curve according to bright scale value (BSV) of CD133 mRNA was used to group patients for prognosis analysis. Results. BSV of preoperative CD133 mRNA in PBMCs in GC was significantly higher than that in volunteers or in GU. Invasive depth or metastatic lymph node number for higher BSV of preoperative CD133 mRNA and invasive depth or lymphatic vessel invasion for higher BSV of postoperative CD133 mRNA in the PBMCs were identified. Patients with CD133+ expression in primary lesion had a significantly higher expression of preoperative CD133 mRNA in the PBMCs. The expression of preoperative or postoperative CD133 mRNA in PBMCs related positively to CD133 mRNA expression in primary lesion. Patients with higher expression of preoperative or postoperative CD133 mRNA shared significantly shorter survival compared with that in lower expression group. Conclusion. Higher levels of preoperative or postoperative CD133 mRNA in PBMCs of GC correlated positively to the lymphatic metastasis and the BSV of CD133 mRNA in primary lesion, indicating the poorer survival.

Imbalanced expression of human Tim-1 and Tim-3 in peripheral blood mononuclear cells from immune thrombocytopenia patients

The T-cell immunoglobulin and mucin domain-(Tim)-1 molecule and Tim-3 are mainly expressed on activated T helper (Th) 2 and Th1 cells, respectively, and have been implicated in the pathogenesis of some autoimmune diseases. Immune thrombocytopenia (ITP) is a common autoimmune disorder, and the complex dysregulation of cellular immunity has been observed; however, the relationship between Tims and excessive immune responses in ITP remains unclear. Using real-time quantitative polymerase chain reaction (RT-PCR), the mRNA expression levels of Tim-1, Tim-3, T-box transcription factor (T-bet) and GATA binding protein 3 (GATA-3) were measured in the peripheral blood mononuclear cells (PBMCs) of 45 newly diagnosed patients with active ITP, 34 ITP patients in remission and 31 healthy volunteers. Tim-3 mRNA expression in PBMCs in newly diagnosed patients was significantly decreased. At the same time, Tim-1 mRNA was not significantly declined, which resulted in a decreased ratio of Tim-3 to Tim-1 in ITP patients with active disease. During the remission stages, the levels of these transcription factors were comparable with those observed in healthy controls. The reduced levels of Tim-3/Tim-1 in PBMCs during active stages of the disease suggest a possible role in the pathogenesis and course of ITP. Regulating the balance of Tim-1 and Tim-3 in ITP patients could also be a therapeutic approach against ITP.

SIRT1 is decreased during relapses in patients with multiple sclerosis

SIRT1 is a member of the histone deacetylase (HDAC) class III family of proteins and is an NAD-dependent histone and protein deacetylase. SIRT1 can induce chromatin silencing through the deacetylation of histones and can modulate cell survival by regulating the transcriptional activities. We investigated the expression of SIRT1 in multiple sclerosis (MS) brains and in peripheral blood mononuclear cells (PBMCs) obtained from patients with relapsing–remitting multiple sclerosis. We found that SIRT1 was expressed by a significant number of cells in both acute and chronic active lesions. We also found that CD4+, CD68+, oligodendrocytes (OLG), and glial fibrillar acidic protein (GFAP)+ cells in MS plaques co-localized with SIRT1. Our results show a statistically significant decrease in SIRT1 mRNA and protein expression in PBMCs during relapses when compared to the levels in controls and stable MS patients. On the other hand, HDAC3 expression was not significantly changed during relapses in MS patients. SIRT1 expression correlated with that of histone H3 lysine 9 acetylation (H3K9ac) and methylation (H3K9me2). SIRT1 mRNA expression was significantly reduced after RGC-32 silencing, indicating a role for RGC-32 in the regulation of SIRT1 expression. Furthermore, we investigated the role of SIRT1 in the expression of FasL and found a significant increase in FasL expression and apoptosis after inhibition of SIRT1 expression. Our data suggest that SIRT1 may represent a biomarker of relapses and a potential new target for therapeutic intervention in MS.

Lysophosphatidylcholine and Carotid Intima-Media Thickness in Young Smokers: A Role for Oxidized LDL-Induced Expression of PBMC Lipoprotein-Associated Phospholipase A2?

BackgroundAlthough cigarette smoking has been associated with carotid intima-media thickness (CIMT) the mechanisms are yet not completely known. Lysophosphatidylcholine (lysoPC), a main product of lipoprotein-associated phospholipase A2 (Lp-PLA2) activity, appears to be a major determinant of the pro-atherogenic properties of oxidized LDL (oxLDL) and to induce proteoglycan synthesis, a main player in intimal thickening. In this study we assessed whether cigarette smoking-induced oxidative stress may influence plasma Lp-PLA2 and lysoPC and Lp-PLA2 expression in peripheral blood mononuclear cells (PBMC), as well as the relationship between lysoPC and CIMT.Methods/Results45 healthy smokers and 45 age and sex-matched subjects participated in this study. Smokers, compared to non-smokers, showed increased plasma concentrations of oxLDL, Lp-PLA2 and lysoPC together with up-regulation of Lp-PLA2 (mRNA and protein) expression in PBMC (P<0.001). Plasma Lp-PLA2 positively correlated with both lysoPC (r=0.639, P<0.001) and PBMC mRNA Lp-PLA2 (r=0.484, P<0.001) in all subjects. Moreover CIMT that was higher in smokers (P<0.001), positively correlated with lysoPC (r=0.55, P<0.001). Then in in vitro study we demonstrated that both oxLDL (at concentrations similar to those found in smoker’s serum) and oxidized phospholipids contained in oxLDL, were able to up-regulate mRNA Lp-PLA2 in PBMC. This effect was likely due, at least in part, to the enrichment in oxidized phospholipids found in PBMC after exposure to oxLDL. Our results also showed that in human aortic smooth muscle cells lysoPC, at concentrations similar to those found in smokers, increased the expression of biglycan and versican, two main proteoglycans.ConclusionsIn smokers a further effect of raised oxidative stress is the up-regulation of Lp-PLA2 expression in PBMC with subsequent increase of plasma Lp-PLA2 and lysoPC. Moreover the correlation between lysoPC and CIMT together with the finding that lysoPC up-regulates proteoglycan synthesis suggests that lysoPC may be a link between smoking and intimal thickening.

Up-regulation of inducible heat shock protein-70 expression in multiple sclerosis patients

Inducible heat shock protein (HSP)70 (HSP70-1A and HSP70-1B proteins) is a chaperone responsible for assisting proper protein folding. Following stress conditions, HSP70 is highly up-regulated to mediate cytoprotective functions. In addition, HSP70 is able to trigger innate and adaptive immune responses that promote the immune recognition of antigens and to act as a cytokine when it is released. The data in the literature are controversial with regard to expression studies in peripheral blood mononuclear cells (PBMCs). In the present study, we aimed to examine if alterations of HSP70-1A/B expression are involved in the autoimmune pathogenesis of multiple sclerosis (MS). We determined both mRNA and protein expression in PBMCs of MS patients and healthy donors (HDs). We found a baseline increased expression of the HSPA1A gene in PBMCs from MS patients compared with HDs. Gene expression findings were associated with an increased protein expression of HSP70-1A/B in T lymphocytes (CD4+ and CD8+) and monocytes from MS patients under basal conditions that may reflect the immunological activation occurring in MS patients. We also provided evidence that heat shock (HS) stimulus induced HSP70-1A/B protein expression in HDs and MS patients, and that HS-induced HSP70-1A/B protein expression in monocytes correlated with the number of T2 lesions at baseline in MS patients. However, after lipopolysaccharide inflammatory stimulus, monocytes from MS patients failed to induce HSP70-1A/B protein expression. Our data hint at altered immune responses in MS and may indicate either a state of chronic stress or increased vulnerability to physiological immune responses in MS patients.

Protein Kinase C-βii-Dependent Up-Regulation Of Heme Oxygenase-1 In Chronic Lymphocytic Leukemia Cells Is Crucial For Resistance Against Imatinib

ObjectiveResistance toward imatinib and other BCR/ABL tyrosine kinase inhibitors(TKI) remains an increasing clinical problem in the treatment of advanced stages of chronic myeloid leukemia (CML). Heme oxygenase-1 (HO-1) inhibition plus TKI were found to produce growth inhibition in imatinib-resistant K562/IM cells and in Ba/F3 cells harboring the T315I mutant of BCR/ABL. In this study, we aimed to explore the molecular mechanism of HO-1 explosive expression and relationship between HO-1 and Protein Kinase C-βII(PKC-βII) in imatinib-resistant CML cells. MethodsWe explored that mRNA expression of various drug-resistant-relative gene between K562 cells and K562/IM cells via high throughout screening assay detected by real-time PCR. According to the results mentioned above, relative protein expression and phosphorylation examined by western blot. And protein translocation was observed and recorded by fluorescence microscope. Cells apoptosis, reactive oxygen species(ROS) and intracellular calcium were analyzed by flow cytometry. Cells proliferation inhibition was detected by CCK-8 assay. The peripheral blood mononuclear cells(PBMNCs) were purified from 45 patients with freshly diagnosed CML or imatinib-resistant CML(including all kinds of drug-resistance involved in various of reasons). Compared the differences of mRNA expression between HO-1 and PKC-βII in those patients. ResultsOur results indicated that PKC-βII contributed to activation of HO-1 expression through PI3K/AKT pathway. PKC-βII phosphorylation localized to membrane from cytoplasim to activate PI3K/AKT pathway, meanwhile, the 7.8 folder higher (p<0.01) expression of HO-1 following the nucleus translocation of NF-E2–related factor 2(Nrf-2) was observed after pAKT up-regulation. The accumulation of ROS and intracellular calcium overload were reduced by HO-1 to protect cells against cytotoxicity, less apoptotic imatinib-resistant K562 cells were detected at the same concentration of imatinib(the total apoptotic rate of K562/IM:42.4%±3.3% vs. K562: 72.1%±4.1%, at the concentration of 1 μM, p<0.01). In addition, the results of HO-1 and PKC-βII mRNA expression in PBMNCs from 45 patients indicated that HO-1 activation kept positively correspondent with expression of PKC-βII in primary leukemic cells obtained from those patients. In addition, blockage of PI3K/AKT, silence of PKC-βII or HO-1 by small interfere RNA( or inhibited by targeted inhibitor) could significantly increase apoptotic rate of imatinib-resistant CML cells(the apoptotic rate of regulated expression/ natural expression: when blockage of PI3K/AKT: 1.54±0.17; silence of PKC-βII: 2.09±0.23; inhibition of HO-1: 1.87±0.12, p<0.05). ConclusionSelectively targeted-PKC-βII plus imatinib produced growth inhibition in primary leukemic cells. In summary, these data showed that HO-1 expression involved in PKC-βII activation in imatinib-treated CML cells, and targeted- PKC-βII may be the same promising novel strategy as HO-1 inhibition in imatinib resistant CML. Disclosures:No relevant conflicts of interest to declare.

KIAA0101 mRNA overexpression in peripheral blood mononuclear cells acts as predictive marker for hepatic cancer

KIAA0101 always overexpresses in tumor tissues, which is also a marker of tumor recurrence. This study aims to explore whether overexpression of KIAA0101 mRNA in peripheral blood mononuclear cells (PBMCs) could act as a noninvasive and predictive biomarker of hepatic cancer. Real-time polymerase chain reaction (RT-PCR) was employed to detect KIAA0101 mRNA expression in PBMCs isolated from 93 hepatic cancer patients and 55 healthy individuals. The diagnostic sensitivity and specificity of KIAA0101 mRNA, CEA, and CD44V were analyzed and compared. A multivariate logistic regression analysis was utilized to analyze risk factors for overall survival of hepatic cancer patients. A concordance analysis was employed to compare the overexpression of KIAA0101 mRNA with clinicopathological diagnosis. All of the 93 hepatic cancer patients were followed up routinely at least 36 months or until death to analyze the 3-year overall survival rate. The results indicated that KIAA0101 mRNA expression was increased significantly in hepatic patients' PBMCs, when compared with that of healthy individuals (P < 0.05). Both the sensitivity and specificity of KIAA0101 mRNA in PBMCs were enhanced significantly compared with those of the CEA and CD44V biomarkers. The multivariate logistic regression analysis indicated that the KIAA0101 mRNA level and pTNM stage were significantly related with the overall survival of the hepatic patients. There was a better concordance between KIAA0101 mRNA overexpression and clinicopathological diagnosis for hepatic cancer (kappa = 0.914, P < 0.001). KIAA0101 mRNA overexpression in PBMCs decreased the 3-year survival rate significantly. In conclusion, overexpression of KIAA0101 mRNA in PBMCs could act as a predictive biomarker for hepatic cancer and has a better sensitivity and specificity.

AB0343 A high serum level of haptoglobin is associated with the response of 12 weeks methotrexate therapy in recent-onset rheumatoid arthritis patients

BackgroundAmong the several available DMARDs, Methotrexate (MTX) is currently the most widely used drug in clinical practice. About 30-40% of the patients could experience an adequate response to MTX alone;...

Faecalibacterium prausnitzii upregulates regulatory T cells and anti-inflammatory cytokines in treating TNBS-induced colitis

Background and aimsFaecalibacterium prausnitzii (F. prausnitzii) is a common anaerobic bacteria colonized in the human gut and inflammatory bowel disease (IBD) patients are usually lack of F. prausnitzii. The aims of this study were to evaluate the anti-inflammatory and immunomodulatory capacity of F. prausnitzii by comparing it with Bifidobacterium longum (B. longum) in both cellular and animal experiments. MethodsHuman peripheral blood mononuclear cells (PBMCs) and 2, 4, 6-trinitrobenzenesulphonic acid (TNBS)-induced colitis rat models were treated with F. prausnitzii, B. longum, F. prausnitzii supernatant or F. prausnitzii medium, respectively. Interleukin (IL)-10, TGF-β1 and IL-12p70 in human PBMCs culture supernatant and rat blood serum were detected. The frequency of CD25+Foxp3+Treg in human PBMCs, rat PBMCs and rat splenocytes were investigated. Besides, the T-bet, GATA-3, ROR-γt and Foxp3 mRNA in human PBMCs, histopathologic characteristics of the intestinal mucosal and weight loss in the rat models were examined. ResultsF. prausnitzii, B. longum and F. prausnitzii supernatant clearly facilitated the induction of IL-10 and TGF-β1, while induced relatively mild production of IL-12p70 in both cellular and animal models. The F. prausnitzii, B. longum and supernatant differed in their capacity to induce T-bet, GATA-3 and ROR-γt mRNA expression in human PBMCs (both bacterial strains inhibited the expression of ROR-γt while supernatant inhibited the T-bet and GATA-3). However, all of them induced the Foxp3 and Treg production and ameliorated the TNBS-induced colitis. In addition, F. prausnitzii supernatant exhibited the supreme anti-inflammatory capacity. ConclusionsF. prausnitzii and its unidentified metabolites in the supernatant are promising candidates in treating IBD, and further research remains necessary to elucidate the safety, efficacy, optimum and mechanism of this bacterium in the clinical practice.

Cytokine gene expression in intestinal tuberculosis and Crohn's disease

Intestinal tuberculosis (TB) and Crohn's disease closely resemble each other clinically and morphologically. Little is known of cytokine regulation in intestinal TB. To compare cytokine gene expression in colonic mucosa and peripheral blood mononuclear cells (PBMC) in TB with that in Crohn's disease. Biopsies were obtained from normal and ulcerated colonic mucosa of 12 intestinal TB and 11 Crohn's disease patients, and PBMC from 15 intestinal TB and 12 Crohn's disease patients and 11 healthy volunteers. RNA was extracted, and the expression of selected cytokines, chemokines and pattern recognition receptors quantified by reverse transcriptase real-time polymerase chain reaction using SYBR green. The mRNA expression of interleukin-8 (IL-8), induced protein-10, tumour necrosis factor-alpha, IL-23 p19 and IL-12 p40, and Toll-like receptors (TLR) 1 and 2 in the ulcerated mucosa was increased in both intestinal TB and Crohn's disease. Expression of growth-related oncogene-alpha was increased in intestinal TB, while expression of interferon-gamma (IFN-) and TLR 4, 5 and 9 was increased in Crohn's disease. Expression of RANTES (regulated upon activation, normal T-cell expressed and secreted) was decreased in Crohn's disease. Secretion of IFN- or IL-10 from PBMC was not significantly altered in either disease. PBMC mRNA expression of IL-1, IL-6 and IL-8 mRNA was upregulated in Crohn's disease, while that of IL-17 was upregulated in intestinal TB. Cytokine gene expression patterns in intestinal mucosa and PBMC of intestinal TB were remarkably similar to Crohn's disease, and demonstrated innate immune activation and T-helper 1 polarisation.

Effects of atorvastatin on CYP3A4 and CYP3A5 mRNA expression in mononuclear cells and CYP3A activity in hypercholeresterolemic patients

BackgroundVariability of response to statins has been related to polymorphisms in genes involved in cholesterol homeostasis and statin metabolism, such as CYP3A4 and CYP3A5. We investigated the effects of atorvastatin on CYP3A4 and CYP3A5 mRNA expression in mononuclear cells and on CYP3A activity and their interactions with common variants. MethodsUnrelated individuals (n=121) with hypercholesterolemia (HC) were treated with atorvastatin (10mg/day/4weeks). Ninety-two normolipidemic (NL) subjects were selected as a control group. Genotype analysis of CYP3A4*1B (rs2740574), CYP3A4*22 (rs35599367), CYP3A5*3C (rs776746), and CYP3A5*1D (rs15524) and mRNA levels in peripheral blood mononuclear cells (PBMCs) were estimated. CYP3A activity was phenotyped by the urinary cortisol to 6-beta-hydroxy-cortisol ratio. ResultsLDL cholesterol reduction in response to atorvastatin was positively correlated with change in CYP3A4 (R2=0.039, p=0.037) and CYP3A5 (R2=0.047, p=0.019) mRNA levels and negatively correlated with CYP3A activity (R2=0.071, p=0.022). CYP3A5*3C (AGT haplotype) was associated to lower basal CYP3A5 mRNA expression in HC (p<0.045), however none of the haplotype groups impacted treatment. ConclusionIt is likely that cholesterolemia status changes promoted by atorvastatin play a role in regulating CYP3A4 and CYP3A5 mRNA expression in PBMCs, as well as CYP3A activity. CYP3A5*3C (AGT haplotype) also contributes for the variability of CYP3A5 mRNA levels in PBMCs.