mRNA In Peripheral Blood Mononuclear Cells Research Articles

Expressions of CD147 in peripheral monocytes and T lymphocytes of patients with ankylosing spondylitis

To investigate the roles of CD147 in the pathogenesis and development of ankylosing spondylitis (AS). Flow cytometry was used to detect the expression levels of CD147 in peripheral monocytes and T lymphocytes of 30 AS patients, 30 rheumatoid arthritis (RA) patients and 30 healthy controls (HC). reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate the expression levels of CD147 mRNA in peripheral blood mononuclear cells (PBMC). Then the expression levels of CD147 were compared among the groups. And a correlation analysis was conducted between CD147 levels and disease activity indices of AS patients. The mean fluorescence intensities of CD147 in monocytes of AS, RA and HC group were 213.5 ± 37.8, 228.7 ± 49.7 and 163.6 ± 44.8, and in T lymphocytes 36.8 ± 10.1, 40.2 ± 10.5 and 28.3 ± 10.6 respectively. Both the expression levels of CD147 in monocytes and T lymphocytes of AS patients were slightly lower than those of RA patients. But the differences was not statistically significant (P > 0.05). Both the CD147 levels in monocytes and T lymphocytes of AS and RA group were significantly higher than those of HC group (P < 0.05). The expression levels of CD147 mRNA in PBMC of AS and RA group were significantly higher than those of HC group (P < 0.05) while no significant difference was found between AS and RA group (P > 0.05). Both the expression levels of CD147 in monocytes and T lymphocytes of AS patients were positively correlated with the patients' erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). The expressions of CD147 in peripheral monocytes and T lymphocytes of AS patients are up-regulated and their levels are positively correlated with patients' ESR and CRP. It implies that CD147 plays critical roles in the pathogenesis and development of AS.

E0102 Expression or secretion of IL-34 and IL-35 in the peripheral blood mononuclear cells from patients with dilated cardiomyopathy

ObjectiveThe aim of this study was to observe the level of interleukin (IL)-34 and IL-35 in peripheral blood mononuclear cells (PBMCs) with dilated cardiomyopathy (DCM), and explore the role of...

Characterized Polysaccharides from Black Soybean Induce Granulocyte Colony-Stimulated Factor Gene Expression in a Phosphoinositide 3-Kinase-dependent Manner

Black soybean (Glycine max L. merr.) is an edible Chinese medicine for nourishment spleen. In the present study, effects of characterized polysaccharides from black soybean (PGM) on granulocyte colony-stimulated factor (G-CSF) production in human peripheral blood mononuclear cells (PBMC) were determined and their action mechanisms were examined. The results indicated that PGM concentration-dependently enhanced G-CSF production in PBMC through modulation of mRNA expression. Data from Western blotting showed that PGM significantly induced the extracellular signal-regulated protein kinase (ERK) activation in PBMC. The nuclear factor (NF)-κB activation in PBMC was increased with PGM by modulation of IκB degradation and PKC θ activation. The levels of G-CSF mRNA in PGM-treated PBMC could be reduced by ERK inhibitor U0126 and NF-κB inhibitor pyrrolidine dithiocarbamate, respectively. Furthermore, the data showed that PGM stimulated phosphoinositide 3-kinase (PI3K)-regulated Akt phosphorylation. The PI3K inhibitor, Ly294002, blocked ERK, NF-κB, and PKC θ activation and G-CSF mRNA expression in PBMC induced by PGM. Thus, we first proved that the enhancement mechanisms of PGM on G-CSF production, appeared to be mediated, at least in part, through activation of PI3K, ERK, PKC θ, and NF-κB signaling pathways in PBMC. We suggest that PGM from black soybean is a potential G-CSF stimulator.

Tumor Necrosis Factor Receptor 1 Expression Is Upregulated in Dendritic Cells in Patients with Chronic HCV Who Respond to Therapy

The present studies assessed the level of tumor necrosis factor receptor (TNFR) expression in peripheral blood mononuclear cells (PBMCs) subsets from patients with chronic HCV undergoing interferon α/ribavirin-based therapy (Ifn/R). Methods. TNFR family member mRNA expression was determined using quantitative real-time PCR assays (RTPCRs) in PBMC from 39 HCV+ patients and 21 control HCV− patients. Further subset analysis of HCV + patients (untreated (U), sustained virological responders (SVR), and nonresponders (NR)/relapsers (Rel)) PBMC was performed via staining with anti-CD123, anti-CD33, anti-TNFR1 or via RTPCR for TNFR1 mRNA. Results. A similar level of TNFR1 mRNA in PBMC from untreated HCV+ genotype 1 patients and controls was noted. TNFR1 and TNFR2 mRNA levels in PBMC from HCV+ patients with SVR were statistically different than levels in HCV(−) patients. A significant difference was noted between the peak values of TNFR1 of the CD123+ PBMC isolated from SVR and the NR/Rel. Conclusion. Upregulation of TNFR1 expression, occurring in a specific subset of CD123+ dendritic cells, appeared in HCV+ patients with SVR.

Responses to surgical stress after esophagectomy: Gene expression of heat shock protein 70, toll-like receptor 4, tumor necrosis factor-α and inducible nitric oxide synthase

The progression and interrelationship of mediators that are released, activated or suppressed after major surgery appear to play an important role in responses to surgical stress. Heat shock protein 70 (HSP70) is stress-induced and acts like a cytokine to modulate pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS), by stimulating toll-like receptor4 (TLR4) signaling. We hypothesized that this effect would occur after major surgery, such as esophagectomy. We therefore measured the expression of HSP70, TLR4, TNF-α and iNOS mRNA in peripheral blood mononuclear cells (PBMCs) from 11 patients who underwent esophagectomy with thoracoabdominal procedures at postoperative day (POD) 1 and POD3 using real-time polymerase chain reaction, and compared the results to expression levels in 6 healthy adult volunteers (controls). We also measured plasma cortisol as a well-known stress hormone. The expression of HSP70 mRNA in PBMCs was 2.1-fold higher on POD1 compared to the controls (P=0.041) and was positively correlated with TLR4 mRNA (r2=0.45, P=0.0007). The expression of TNF-α mRNA tended to be lower on POD1 (P=0.055) and was significantly decreased on POD3 (P=0.016), and iNOS mRNA were significantly lower on POD1 (P=0.0015) and POD3 (P=0.0003) compared to the controls. Moreover, there was a positive correlation between the expression of TLR4 mRNA and plasma cortisol levels (r2=0.24, P=0.021). The expression of HSP70 mRNA in PBMCs in the early postoperative period was significantly higher and positively correlated with TLR4 mRNA. This suggests that HSP70-TLR4 signaling has an important role in postoperative inflammatory responses. However, the expression of pro-inflammatory mediators, including TNF-α and iNOS mRNA, was significantly decreased postoperatively. This may be caused by the anti-inflammatory mechanism of cortisol. Our findings indicate that responses to surgical stress reflect simultaneous pro-inflammatory and anti-inflammatory responses, and are complex.

Increased Expression of Suppressor of Cytokine Signaling 1 mRNA in Patients With Rheumatoid Arthritis

The objective of this study was to investigate the associations between suppressor of cytokine signaling 1 (SOCS1) mRNA expression and SOCS1 polymorphisms with the development of rheumatoid arthritis (RA). One hundred and eighty-one patients with RA and 96 healthy controls were enrolled in this study. The SOCS1 mRNA level in peripheral blood mononuclear cells (PBMCs) was detected by quantitative real-time polymerase chain reaction. SOCS1 polymorphisms were determined by the polymerase chain reaction/restriction fragment length polymorphism method. We found that the expression of SOCS1 mRNA in PBMCs was significantly greater in patients with RA than in healthy controls. There were no significant differences in the expression of SOCS1 mRNA among patients with different disease activities. The increment in SOCS1 mRNA after stimulation with various cytokines was slightly lower in the patients with RA than in the healthy controls. This study also demonstrated that the SOCS1 polymorphisms were not associated with susceptibility to RA. In conclusion, the expression of SOCS1 mRNA in PBMCs is higher in patients with RA than in healthy controls. The increment in SOCS1 mRNA expression in PBMCs after stimulation with different cytokines seems to be lower in patients with RA than in healthy controls.

Differentiation Imbalance of Th1/ Th17 in Peripheral Blood Mononuclear Cells Might Contribute to Pathogenesis of Hashimoto’s Thyroiditis

T helper 17(Th17) cell is a new subset of CD4(+) T cells that produce a proinflammatory cytokine interleukin-17 (IL-17). Th17 cells have recently been shown to play a critical role in many autoimmune diseases that had previously been thought to be Th1 dominant. Although Hashimoto's thyroiditis (HT) was thought to be a Th1-type disease, the contributions of Th17 cells to the pathogenesis remain unclear. In this study, we investigated the expression levels of Th1/Th17 cell-associated factors in peripheral blood mononuclear cells (PBMC) and plasma from patients with HT by quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Our results showed that the expression levels of Th1 cells-related T-bet and interferon-gamma (IFN-gamma) mRNA in PBMC from HT significantly decreased. However, the mRNA of Th17 coherent retinoic acid-related orphan nuclear receptor gamma t (RORgammat) and IL-17 in patients with HT increased. In addition, a negative correlation between T-bet and RORgammat mRNA expression was found in patients with HT, and the similar phenomena also appeared on the levels of mRNA and plasma concentration between IFN-gamma and IL-17. It suggested that Th17 cells rather than Th1 cells predominated among patients suffering from HT, and Th17 cells might be involved in the pathogenesis of HT.

A pilot study of mRNA expressions of 5-fluorouracil pathway genes in peripheral blood mononuclear cells and tumor tissues in patients with lung adenocarcinoma

To assess whether early lung cancer prediction might be informed by an mRNA assay for 5-fluorouracil pathway genes in peripheral blood mononuclear cells (PBMNCs), we examined specimens taken from 51 adenocarcinoma patients and 38 controls (including six patients with benign tumors). PBMNCs and tumor-tissue specimens were taken for measurement of the mRNAs of various 5-fluorouracil pathway genes [thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), and orotate phosphoribosyl transferase (OPRT)]. By quantitative RT-PCR, all four mRNAs were detected in both PBMNCs and tumor tissues. In PBMNCs, TS mRNA/GAPDH mRNA levels were significantly higher in adenocarcinoma patients than in the controls, and significantly higher for pathological stages 2–4 and lymph-node involvement pN1–pN3 than for pathological stage 1 and pN0, respectively. No correlation between PBMNCs and tumor-tissue specimens was found for the level of any mRNA. Thus, the measurement of TS mRNA in PBMNCs might aid the diagnosis of lung adenocarcinoma.

Low responder T cell susceptibility to the suppressive function of regulatory T cells in patients with dilated cardiomyopathy

ObjectiveThe pathogenesis of dilated cardiomyopathy (DCM) is closely connected with dysfunction of the autoimmune system, and CD4+CD25highCD127low/− regulatory T (Treg) cells have a vital role in maintaining self-tolerance. In this...

Effects of Cordyceps extract on cytokines and transcription factors in peripheral blood mononuclear cells of asthmatic children during remission stage

To explore the effects of Cordyceps extract in regulating the imbalance of Th1/Th2 ratio and inhibiting the inflammatory reaction, and to find the theoretical basis of Cordyceps extract for treating asthma in remission stage. A total of 20 peripheral venous blood samples (3 mL) were collected from 20 asthmatic children during remission stage, and peripheral blood mononuclear cells (PBMCs) were separated by Ficoll method. PBMCs were separated into three groups (blank group, low-dose group and high-dose group). The PBMCs were incubated in vitro for 48 hours in the absence (blank group) or presence (low-dose group and high-dose group) of Cordyceps extract at different concentrations (10, 20 microg/mL). The expressions of interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-10, T-box expressed in T cells (T-bet), GATA-binding protein-3 (GATA-3) and forkhead/winged-helix transcription factor-3 (Foxp3) mRNAs in PBMCs were measured by real-time fluorescent quantitative polymerase chain reaction, and the contents of IFN-gamma, IL-4 and IL-10 in supernatants were measured by enzyme-linked immunosorbent assay. The expressions of IFN-gamma mRNA showed no significant differences among the three groups. The expressions of the IL-4 mRNA in the high-dose group and the low-dose group were lower than that in the blank group (P=0.014, P=0.011). The expression of IL-10 mRNA in the high-dose group was higher than that in the blank group (P=0.034). And the differences of the IFN-gamma mRNA/IL-4 mRNA ratio presented no statistic significance among the three groups. The level of IL-4 content in the high-dose group was lower than that in the blank group (P=0.018), but the level of IL-10 content, and ratio of IFN-gamma/IL-4 in the high-dose group were higher than those in the blank group (P=0.011, P=0.045). The differences of the IFN-gamma presented no statistic significance among the three groups. The T-bet/GATA-3 ratio and Foxp3 mRNA expression in the high-dose group were higher than those in the blank group (P=0.001, P=0.015). There was significant difference in expression of GATA-3 mRNA between the high-dose group and the blank group (P=0.028), and between the low-dose group and the blank group (P=0.019). The expression differences of T-bet mRNA were insignificant between any two groups. Cordyceps extract can inhibit the proliferation and differentiation of Th2 cells and reduce the expression of related cytokines by down-regulating the expression of GATA-3 mRNA and up-regulating the expression of Foxp3 mRNA in PBMCs. Meanwhile, it can alleviate the chronic allergic inflammation by increasing the content of IL-10.

Effects of selenium source on blood selenium content, blood cell counts and peripheral blood mononuclear cell mRNA profiles in maturing beef heifers

To determine if source of dietary selenium (Se) supplementation affects blood Se content and expression of biomarkers of immune status in blood cells of growing cattle, Angus heifers were assigned (n = 10) to 4 Se treatments (TRT) for 126 d. The basal diet contained 0.08 mg Se/d, whereas the mineral supplements provided no (control, C), or 3 mg Se/d as inorganic (sodium selenite, SS), organic (Sel‐Plex®, SP), or a 50/50 mix (SS/SP). Jugular blood was collected for blood cell counts, Se analysis and peripheral blood mononuclear cell (PBMC) isolation. Whole blood (WB), plasma, and red blood cell (RBC) Se content was greater (P &lt; 0.01) for all TRT versus C. SS/SP and SP heifers had greater (P &lt; 0.01) WB and RBC Se content than SS, while plasma Se tended (P = 0.06) to be higher in SS/SP heifers than SS. PBMC count was negatively correlated (P &lt; 0.05) to plasma and WB Se content in SP heifers, and tended (P = 0.06) to be negatively correlated to WB Se in SS/SP heifers. Se TRT did not affect interleukins 1β, 8, and receptor 2α mRNA levels in PBMC, while selenoprotein S (SelS) mRNA content was decreased (P &lt; 0.05) in SS/SP and SP versus C heifers. Because SelS plays an important role in the regulation of the inflammatory cytokine response, decreased SelS mRNA levels indicate supplementing with 3 mg/d SP or SS/SP may result in cattle with a less challenged immune state. This work is supported by the Alltech‐University of KY Animal Nutrigenomics Alliance.

Expression of Th1 and Th2 cytokine-associated transcription factors, T-bet and GATA-3, in peripheral blood mononuclear cells and skin lesions of patients with psoriasis vulgaris

Psoriasis is considered as a T cell-mediated autoimmune disease, and the Th1 response has been established as the major immune agent in its pathomechanisms. The relative expression of Th1 and Th2 transcription factors, T-bet and GATA-3, resulting in a swing in the Th1/Th2 pendulum, has been implicated in a number of immunological diseases. However, their expression and correlation with psoriasis has not yet been studied. Our aim was to evaluate the expression of T-bet and GATA-3 in psoriatic patients and determine their correlation with serum levels of IFN-gamma and IL-4. Sera, peripheral blood mononuclear cells (PBMCs) and skin lesions were taken from 23 patients with psoriasis vulgaris. Serum levels of IFN-gamma and IL-4 were measured by ELISA. T-bet and GATA-3 mRNA expression in PBMCs was analyzed by RT-PCR. Lesional expression and distribution of CD4, CD8, T-bet and GATA-3 were assessed by immunohistochemistry. Blood and skin samples of healthy individuals served as controls. A markedly higher IFN-gamma and lower IL-4 concentration in the serum of psoriatic patients was found. A significantly higher expression of T-bet mRNA and a lower expression of GATA-3 mRNA in PBMCs, and consequently, a much higher T-bet/GATA-3 ratio in patients than in controls were shown. T-bet mRNA expression was strongly correlated with serum IFN-gamma secretion in patients; furthermore, the correlation between T-bet/GATA-3 ratio and IFN-gamma/IL-4 ratio was revealed. We also observed a significant increase in CD4+ cells and T-bet positive cells in psoriatic lesions. These results suggested that T-bet and GATA-3 might be regulator genes for psoriasis via the Th1/Th2 balance, and the Th1-specific transcription factor, T-bet, may play an important role in the development and maintenance of psoriasis.

Can blood components with age‐related changes influence the ageing of endothelial cells?

Research on vascular endothelial cell ageing helps elucidate the pathogenesis of diseases associated with cell ageing. To investigate endothelial senescence, we used 2-DE coupled with MS to perform a proteomic analysis of: (i) peripheral blood mononuclear cells (PBMCs) from donors in their 20 s ('young') or 60 s ('old') and (ii) human dermal microvascular endothelial cells (HDMECs) treated with sera from young and old donors. Identified proteins could be classified into several functional categories: (i) cytoskeletal regulators: CapG and cofilin 1; (ii) stress response and signal pathway proteins: TXNDC5 and RSU-1; and (iii) apoptosis proteins: Annexin V. We confirmed by Western blot a decrease of RSU-1, CapG and TXNDC5 in PBMCs from old donors. RSU-1, which regulates signal transduction of the downstream Ras, showed decreased mRNA and protein levels in PBMCs from old donors and decreased mRNA levels in HDMECs treated with sera from old donors. In addition, Ras protein levels were increased in PBMCs from old donors. These data indicate that reduced RSU-1 might induce Ras expression, which subsequently could provoke Ras-induced senescence. In conclusion, our data suggest that blood components that exhibit age-related changes, such as alterations in cytoskeletal regulators and stress proteins, may be associated with endothelial cell ageing.

Diagnostic value of cancer-testis antigen mRNA in peripheral blood from hepatocellular carcinoma patients

To evaluate the diagnostic value of cancer-testis antigen (CTA) mRNA in peripheral blood samples from hepatocellular carcinoma (HCC) patients. Peripheral blood samples were taken from 90 patients with HCC before operation. Expression of melanoma antigen-1 (MAGE-1), synovial sarcoma X breakpoint-1 (SSX-1), and cancer-testis-associated protein of 11 kDa (CTp11) mRNA in peripheral blood mononuclear cells (PBMC) was tested by nested reverse transcripts-polymerase chain reaction (RT-PCR). Serum alpha-fetoprotein (AFP) in these patients was also determined. The positive rate of MAGE-1, SSX-1 and CTp11 transcripts was 37.7%, 34.4%, 31.1% in PBMC samples, and 74.4%, 73.3%, 62.2% in their resected tumor samples, respectively. The positive rate for at least one of the transcripts of three CTA genes was 66.7% in PBMC samples and 91.1% in their resected tumor samples. MAGE-1, SSX-1 and/or CTp11 mRNA were not detected in the PBMC of those patients from whom the resected tumor samples were MAGE-1, SSX-1 and/or CTp11 mRNA negative, nor in the PBMC samples from 20 healthy donors and 10 cirrhotic patients. Among the 90 patients, the serum AFP in 44 patients met the general diagnostic standard (AFP > 400 microg/L) for HCC, and was negative (AFP < or = 20 microg/L) or positive with a low concentration (20 microg/L < AFP < or = 400 microg/L) in the other patients. The positive rate for at least one of the transcripts of three CTA genes in PBMC samples from the AFP negative or positive patients with a low concentration was 69.2% and 45.0%, respectively. Of the 90 patients, 71 (78.9%) were diagnosed as HCC by nested RT-PCR and serum AFP. Although the positive rate for at least one of the transcripts of three CTA genes in PBMC samples from 53 patients at TNM stage III or IV was obviously higher than that in PBMC samples from 37 patients at stage I or II (77.9% vs 51.4%, P = 0.010), the CTA mRNA was detected in 41.7% and 56.0% of PBMC samples from HCC patients at stages I and II, respectively. Detecting MAGE-1, SSX-1 and CTp11 mRNA in PBMC improves the total diagnostic rate of HCC.

Serum and Gene Expression Profile of Tumor Necrosis Factor-α and Interleukin-6 in Hypertensive Diabetic Patients: Effect of Amlodipine Administration

Pro-inflammatory cytokines are critical factors in type 2 diabetes-associated atherosclerosis. We aim to analyze in hypertensive type 2 diabetic patients the serum concentrations and the mRNA expression levels in peripheral blood mononuclear cells (PBMC) of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), as well as to evaluate the effect of amlodipine administration. Twenty-one hypertensive diabetic patients and 10 healthy non-diabetic controls were included in the study. Serum levels of cytokines were measured by chemiluminescent immunometric assay, and mRNA expression levels by RT-PCR. The mean serum concentrations of TNF-alpha and IL-6 in diabetic patients showed a 6.1-fold and 2.9-fold increase with respect to non-diabetic control subjects, respectively (p less than 0.0001). Likewise, there was a 3.3- and a 4-fold increase in the PBMC mRNA expression level of TNF-alpha and IL-6 (p less than 0.0001) in diabetic subjects. After amlodipine administration, a significant decrease (p less than 0.01) was observed in the serum TNF-alpha and IL-6 levels. In addition, pre-treatment mRNA expression of TNF-alpha and IL-6 also decreased, with a mean percent reduction of 26 percent (p less than0.01) and 25 percent (p less than 0.001), respectively. In conclusion, serum concentrations and PBMC mRNA expression levels of TNF-alpha and IL-6 are significantly elevated in hypertensive type 2 diabetic patients. Administration of amlodipine is associated with a significant reduction of the increased levels of these inflammatory parameters, both at the protein as well as at the transcriptional level. These modulatory effects of amlodipine on proinflammatory cytokine level and expression may be related to its suggested anti-atherosclerotic actions.

Phase I trial of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a heat shock protein inhibitor, administered twice weekly in patients with advanced malignancies

PurposePhase I dose-escalation study to determine the toxicity and maximum tolerated dose (MTD) of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a heat shock protein 90 (Hsp90) inhibitor, administered on a twice weekly schedule in patients with advanced cancer. Experimental design17-DMAG was administered as a 1- to 2-h infusion twice weekly in 4-week cycles. An accelerated titration design was followed until toxicity was observed, at which point standard dose-escalation proceeded. MTD was defined as the dose at which no more than one of the six patients experienced a dose-limiting toxicity (DLT). Pharmacokinetics were assessed, and Hsp70 mRNA, whose gene product is a chaperone previously shown to be upregulated following the inhibition of Hsp90, was measured in peripheral blood mononuclear cells (PBMCs). ResultsA total of 31 patients received 92 courses of treatment. The MTD was 21mg/m2/d; 20 patients were enrolled at this dose level. Nine patients had stable disease for a median of 4 (range 2–22) months. Both Cmax and AUC increased proportionally with dose. The most common toxicities were grade 1 or 2 fatigue, anorexia, nausea, blurred vision and musculoskeletal pain. DLTs were peripheral neuropathy and renal dysfunction. Expression of Hsp70 mRNA in PBMCs was highly variable. ConclusionTwice-weekly i.v. infusion of 17-DMAG is well tolerated, and combination phase I studies are warranted.

TNF-α and Its Receptors in Patients with Acute Myeloid Leukemia Undergoing alloHSCT.

Abstract 4484 IntroductionSeveral studies have demonstrated, that TNF α and its receptors (sTNFR) can be used as prognostic markers for major transplant related complications (TRC) after allogeneic haematopoietic stem cell transplantation (alloHSCT). Measurement of that inflammatory cytokine and sTNFR during pretransplant conditioning and early after transplantation may also reflect the conditioning-induced tissue damage. Patients and methodsOur study included a group of 36 adult patients with acute myeloid leukemia (AML) in complete remission, who underwent alloHSCT following standard myeloablative conditioning according to Bu/Cy protocol (17 patients) or reduced-toxicity myeloablative conditioning with treosulfan-based regimens (19 patients). 21 patients received alloHSCT from a sibling and 15 from an unrelated donor. The expression of TNF-α, TNFRI and TNFRII were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) in peripheral blood mononuclear cells (PBMC) before the start of conditioning, on the day of transplantation and on day 30 after alloHSCT. We examined β-actin as well, which constitutes an endogenous amplification control and is a housekeeping gene. sTNFRI and sTNFRII serum levels were measured using an enzyme-linked immunosorbent assay (ELISA) at the same defined time points before and after alloHSCT. The Mann-Whitney U test was used to evaluate the significance of differences between the analyzed groups and the Shapiro-Wilk and Lilliefords tests to estimate data distribution. ResultsTNFRII mRNA was not detected in PBMC, but TNF-α and TNFRI mRNA as well as β-actin were discovered. The real time PCR showed significant decrease (p<0.004) in TNFRI expression on day 30 after transplantation in comparison to day 0. On day 30 expression of TNFRI was higher in patients treated with Treosulfan-based regimens than in patients treated with Bu/Cy (p=0.027). Plasma levels of sTNFRI and sTNFRII observed on day 0 and 30 were significantly elevated (p<0.05) compared with the levels prior to conditioning. ConclusionOur data indicate that sTNFRI and sTNFRII can be investigated as the markers reflecting the toxicity of the conditioning regimens in AML patients undergoing alloHSCT. The associations between TNF-α, TNFRI expression, serum levels of sTNFR and the clinical outcome after alloHSCT should be evaluated. Disclosures:No relevant conflicts of interest to declare.

Transforming growth factor-β and Th17 responses in resistance to primary murine schistosomiasis mansoni

Discovery of the T-helper (Th) 17 cell lineage and functions in immune responses of mouse and man prompted us to investigate the role of transforming growth factor-beta (TGF-β) and interleukin (IL)-17 in innate resistance to murine schistosomiasis mansoni. Schistosoma mansoni-infected BALB/c and C57BL/6 mice were administered with recombinant TGF-β or mouse monoclonal antibody to TGF-β to evaluate the impact of this cytokine on host immune responses against lung-stage schistosomula, and subsequent effects on adult worm parameters. Developing schistosomula elicited increase in peripheral blood mononuclear cells (PBMC) mRNA expression and/or plasma levels of IL-4, IL-17, and interferon-gamma (IFN-γ), cytokines known to antagonize each other, resulting in impaired Th1/Th2, and Th17 immune responses and parasite evasion. Mice treated with TGF-β showed elevated PBMC mRNA expression of IL-6, IL-17, TGF-β, and TNF-α mRNA and increased IL-23 and IL-17 or TGF-β plasma levels, associated with significantly ( P < 0.02–<0.0001) lower S. mansoni adult worm burden compared to controls in both mouse strains, thus suggesting that TGF-β led to heightened Th17 responses that mediated resistance to the infection. Mice treated with antibody to TGF-β showed increase in PBMC mRNA expression and plasma levels of IL-4, IL-12p70, and IFN-γ, and significantly ( P < 0.02 and <0.0001) reduced worm burden and liver worm egg counts than untreated mice, indicating that Th1/Th2 immune responses were potentiated, resulting in significant innate resistance to schistosomiasis. The implications of these observations for schistosome immune evasion and vaccination were discussed.

The CGGGG insertion/deletion polymorphism of the IRF5 promoter is a strong risk factor for primary Sjögren's syndrome

Interferon regulatory factor 5 is a transcription factor involved in type I interferon (IFN) secretion. This study was undertaken to investigate whether a 5-bp (CGGGG insertion/deletion) promoter polymorphism is involved in genetic predisposition to primary Sjögren's syndrome (SS) and to assess the functional consequences of this polymorphism. The exploratory cohort consisted of 185 patients with primary SS and 157 healthy controls, and the replication cohort consisted of 200 patients with primary SS and 282 healthy controls. Levels of IRF5 messenger RNA (mRNA) were assessed at baseline and after in vitro infection with reovirus in peripheral blood mononuclear cells (PBMCs) from 30 patients with primary SS and from salivary gland epithelial cells that had been cultured for 4 weeks from patients with primary SS or sicca symptoms. Carriage of the IRF5 4R CGGGG allele was associated with a greatly increased risk of primary SS in both cohorts (odds ratio 2.00 [95% confidence interval 1.5-2.7], P = 6.6 x 10(-6)). The CGGGG insertion/deletion polymorphism alone was sufficient to explain the association of primary SS with IRF5. The level of IRF5 mRNA in PBMCs depended significantly on genotype (P = 0.002) and was correlated with the levels of mRNA for the IFN-induced genes MX1 and IFITM1. Cultured salivary gland epithelial cells from patients carrying the 4R CGGGG IRF5 allele showed a high level of IRF5 mRNA (P = 0.04), which was amplified after reovirus infection (P = 0.026). Our findings indicate an association of the CGGGG insertion/deletion polymorphism of the IRF5 promoter with primary SS. Patients carrying the 4R CGGGG IRF5 allele had a high level of mRNA for IRF5 in PBMCs and salivary gland epithelial cells, mainly after in vitro viral infection. Patients with high levels of mRNA for IRF5 also had high levels of mRNA for type I IFN-induced genes in PBMCs.

Change of Peripheral Blood Mononuclear Cells IFN-γ, IL-10, and TGF-β1 mRNA Expression Levels With Active Human Cytomegalovirus Infection in Orthotopic Liver Transplantation

Aim To analyze the expression levels of interferon gamma (IFN-γ), interleukin 10 (IL-10), and transforming growth factor beta 1 (TGF-β1) mRNA in peripheral blood mononuclear cells (PBMCs) in liver transplanted recipients with active HCMV infection. Methods PBMCs were isolated from 20 liver transplanted recipients with active HCMV infection and 20 recipients without HCMV infection. The expression levels of IFN-γ, IL-10 and TGF-β1 mRNA in PBMCs were measured by TaqMan real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The results were compared with that from 20 healthy individuals. Results The expression level of TGF-β1 mRNA was significantly increased in the active HCMV infection group compared with that in stable group or healthy group ( P < .001). The expression level of IL-10 mRNA was significantly increased in the active HCMV infection group compared with the healthy group ( P = .001). However, the IFN-γ mRNA expression level was significantly decreased in the active HCMV infection group compared with that in the stable group or the healthy group ( P < .05). Conclusion Cytokine production plays a role in the HCMV infection. This may provide an important clue to a better understanding of the pathogenesis in liver transplanted recipients with active HCMV infection.