Peripheral Blood mRNA Research Articles

165 THE EFFECT OF shRNA TARGETING CLUSTER OF DIFFERENTIATION ANTIGEN 14 ON GENE EXPRESSION OF TNF-α, TLR4, AND IL-6 IN LIPOPOLYSACCHARIDE-INDUCED BUFFALO PERIPHERAL BLOOD MONOCYTE/MACROPHAGE

Cluster of differentiation antigen 14 (CD14) plays a crucial role in the inflammatory response to lipopolysaccharide (LPS), which interacts with TLR4 and MD-2 to enable cell activation, leading to inflammation. Several studies have proved that upstream inhibition of bacterial LPS/toll-like receptor 4 (TLR4)/CD14-mediated inflammation pathway is an effective therapeutic approach for attenuating damaging immune activation. In this study, to explore the effect of CD14 down-regulation on TLR4 signal conductive-related genes expression after stimulation by LPS, five CD14 shRNA (319/421/755/970/1041) sequences and a negative control sequence (NC-1864) were synthesised and used to construct lentiviral recombinant plasmid pSicoR-GFP-shRNA. Lentiviral recombinant plasmids of pSicoR-GFP-shRNA and fusion expression vector of pDsRed-N1-buffalo CD14 were co-transfected into HEK293 using liposome. At 72 h after transfection, the expression of exogenous buffalo CD14 mRNA was reduced at different level for all shRNA plasmids, in which shRNA-1041 had the highest interfering efficiency by RT-qPCR and fluorescence-activated cell sorting analysis. Then, buffalo peripheral blood monocyte/macrophage was purified and infected by the CD14 shRNA lentivirus. After 7 days of infection, the cells were stimulated by 1 µg mL–1 LPS for 3 h, then the mRNA expression level of CD14, TLR4, IL-6, and TNF-α transcripts in the cells were detected by the RT-qPCR method. After stimulation by LPS, the expression of endogenous CD14 was significantly reduced by CD14 shRNA-1041, the mRNA expression level of TLR4, IL-6, and TNF-α genes was also significantly down-regulated in comparison with control group (P ≤ 0.01). In conclusion, the selected CD14 shRNA-1041 cannot only inhibit the expression of endogenous CD14 mRNA in buffalo peripheral blood monocyte/macrophage, but also downregulate the mRNA expression of CD14, TLR4, IL-6, and TNF-α. The above results demonstrate that knockdown of endogenous CD14 has obvious coordination effects on the signal conductive function of TLR4 after stimulating by LPS, and shRNA technology will provide a new way to prevent endotoxin-related diseases in livestock. This work was supported by the National Transgenic Project (2009ZX08007-009B), Guangxi natural science funding (2012GXNSFCB053002), and funding of State Key Laboratory of Subtropical Bioresource Conservation and Utilisation (KSL-CUSAb-2012-02).

A Biomarker Obtained From Whole Peripheral Blood Predicts Response to Bortezomib: Results of a Multicenter Prospective Clinical Study of Multiple Myeloma in Japan.

AbstractAbstract 2954 Introduction:The first-in-class proteasome inhibitor bortezomib (Bzb) has revolutionized the treatment of multiple myeloma (MM). However, identifying a biomarker to predict which patients will respond to Bzb is a priority in selecting this treatment or determining whether this powerful but toxic armament should be continued in individual patients. Furthermore, to monitor the efficacy of Bzb, peripheral blood (PB) biomarkers are more desirable than painful bone marrow aspirations. Methods:We conducted a multicenter prospective study to seek biomarkers that predict a response to Bzb. Patients with previously treated or untreated symptomatic MM received twice-weekly or weekly doses of 1.3 mg/m2 Bzb with or without dexamethasone. All patients had measurable M-protein levels. All patients provided written informed consent. Review boards at each site approved the study, which was conducted in accordance with the Declaration of Helsinki. A 2 ml sample of heparinized whole blood was obtained before treatment, as well as 2–3 days and 1–2 weeks after i.v. administration of the first dose of Bzb treatment in the 1st cycle. Triplicate aliquots of 0.06 ml of whole blood were mixed with phytohemaglutinin (PHA), heat-aggregated IgG (HAG), lipopolysaccharide (LPS), zymosan A (ZA), or solvent phosphate buffered saline (PBS) and incubated for 4 hours at 37°C. After incubation, the samples were stored at −80°C. The levels of the target mRNAs (IL2, IFNγ, GMCSF, TNFα, CCL4, IL6, CXCL10, and control ACTB) were quantified from each aliquot using a previously described method (J Immunol Methods 363: 95, 2010). The analysis of mRNA and collection of clinical data were performed separately at different centers. Clinical response was assessed according to the International Uniform Response Criteria. Results:Between March 2010 and March 2012, a total of 64 patients (33 male and 31 female) were enrolled from 6 centers. The median age of all patients was 62 years. The original protocol stipulated that only relapsed or refractory patients were eligible, but in October 2011, the protocol was amended to include newly diagnosed patients due to changes in the Japanese National Health Insurance policy. Consequently, 42 patients were previously treated and 22 patients were untreated. After excluding 1 patient who died early from progressive disease and 1 who received additional treatment, 62 patients were eligible for response analysis. Five patients achieved CR and VGPR, 25 patients obtained PR and SD, and 2 cases of PD were documented. The median follow-up time of estimable patients was 160.5 (range, 26 to 666) days. A total of 2,444 (64 [patients] × 5 [stimulants] × 3 [triplicate] × 3 [time points] with some sampling failure subtracted) mRNA/cDNA samples were prepared, and 19,552 (2,444 [cDNA] × 8 [mRNAs]) PCRs were performed. In total, 53 patient blood samples qualified for mRNA analysis. Among the 32 sample dataset (4 stimulants × 8 mRNAs) obtained before Bzb treatment, LPS-induced CXCL10 correlated with clinical responses, where 9 out of 10 cases of CR and VGPR showed a >=3 fold increase (FI), and both cases of PD showed an FI <3. The FI of PR and SD was distributed widely without a clear distinction. Moreover, after administration of Bzb, LPS-induced CXCL10 declined to less than 30% in all cases of CR and VGPR, whereas PR and SD had mixed results. Interestingly, 7 out of 10 CR and VGPR cases maintained the suppression of LPS-induced CXCL10, even after 1–2 weeks. Conclusion:LPS-induced CXCL10 mRNA in peripheral whole blood is a promising biomarker for the prediction and monitoring of CR and VGPR responses to Bzb treatment. Disclosures:Mitsuhashi:the company who owns the technology of mRNA analysis used in this study: Employment. Abe:Janssen Pharmaceutical K.K.: Honoraria, Research Funding. Nakagawa:Janssen Pharmaceutical K.K.: Honoraria. Nakamura:Janssen Pharmaceutical K.K.: Honoraria. Iida:Janssen Pharmaceutical K.K.: Honoraria. Kizaki:Janssen Pharmaceutical K.K.: Honoraria.

Vascular endothelial growth factor receptor-1 mRNA overexpression in peripheral blood as a useful prognostic marker in breast cancer

IntroductionIdentification of useful markers associated with poor prognosis in breast cancer patients is critically needed. We previously showed that expression of vascular endothelial growth factor receptor-1 mRNA in peripheral blood may be useful to predict distant metastasis in gastric cancer patients. However, expression of vascular endothelial growth factor receptor-1 mRNA in peripheral blood of breast cancer patients has not yet been studied.MethodsReal-time reverse transcriptase-PCR was used to analyze vascular endothelial growth factor receptor-1 mRNA expression status with respect to various clinical parameters in 515 patients with breast cancer and 25 controls.ResultsExpression of vascular endothelial growth factor receptor-1 mRNA in peripheral blood was higher in breast cancer patients than in controls. Increased vascular endothelial growth factor receptor-1 mRNA expression was associated with large tumor size, lymph node metastasis and clinical stage. Patients with high vascular endothelial growth factor receptor-1 mRNA expression also experienced a poorer survival rate than those with low expression levels, including those patients with triple-negative type and luminal-HER2(-) type disease.ConclusionsExpression of vascular endothelial growth factor receptor-1 mRNA in peripheral blood may be useful for prediction of poor prognosis in breast cancer, especially in patients with triple-negative type and luminal-HER2(-) type disease.

The differences and correlations of BCR‐ABL transcripts between peripheral blood and bone marrow assays are associated with the molecular responses in the bone marrow for chronic myelogenous leukemia

Previous studies concerning BCR-ABL mRNA levels by quantitative real-time RT-PCR (Q-PCR) for chronic myelogenous leukemia (CML) have shown a significant concordance between peripheral blood (PB) and bone marrow (BM) assays. The objective of this study was to determine whether molecular monitoring using PB was comparable to using BM for CML. A comparative study was performed that analyzed the Q-PCR results of 712 simultaneous PB and BM samples from 330 patients before and during imatinib therapy. For the 78 paired pretreatment samples, the level of BCR-ABL mRNA in PB was lower than that in BM (P = 0.007). Although the overall amounts of BCR-ABL mRNA in the PB and BM were comparable (P= 0.072) and there was a strong correlation (r = 0.839, P < 0.001) with the 634 paired on-treatment samples, the depth of the molecular response in PB was lower than that in BM (P < 0.001). The level of BCR-ABL mRNA in PB was lower than that in BM where the BM BCR-ABL mRNA < 1 log reduction (P < 0.001) or ≥ 1-< 2 log-reductions (P = 0.008) from the baseline, and higher than that where the BM BCR-ABL mRNA ≥ 2 log-reductions (P < 0.001). A strong correlation (r = 0.811, P < 0.001) was only found where the BM BCR-ABL mRNA < 1 log reduction. We conclude that the differences and correlations of BCR-ABL mRNA between PB and BM assays depend on the depth of the molecular response in BM for CML during imatinib therapy.

Association between Rho-kinase (ROCK2) gene polymorphisms and Behçet’s disease

Behçet's disease (BD) is a multi-systemic vasculitis. The aim of this study was to investigate the association between Rho-kinase (ROCK2) gene polymorphisms and patients with BD in a Turkish population. A total of 194 BD patients and 276 healthy controls with similar age and sex were included to this study. Polymorphisms were analyzed in genomic DNA using a BioMark 96.96 dynamic array system. mRNA from blood samples was extracted, and real-time polymerase chain reaction was performed for ROCK2 gene expression. There were marked changes in both genotype (TT, 41.8%; TA, 30.3%) and allele (T, 57%; A, 43%) frequencies for the rs35768389 (Asp601Val) polymorphism in patients compared with controls (TT, 64.6%; TA, 9.4%, P < 0.0001; T, 69.3%; A, 30.7%, P = 0.0004). Although CC genotype (52.0%) of rs1515219 polymorphism were more frequent, CT genotype (27.7%) were less frequent among the patients than controls (CC, 31.7%, CT, 44.6%, P = 0.0001). There was an increase in C allele (65.8% vs 54.0%) and decrease in T allele frequencies (34.2% vs 46.0%, P = 0.001) in patients. However, no associations were found with rs726843, rs2290156, rs965665, rs10178332, rs2230774, rs6755196, rs10929732, and rs34945852 polymorphisms. There was an increase in peripheral blood mRNA ROCK2 expressions in patients. This is the first study to examine the involvement of ROCK2 gene variation in the risk of incident BD. The results strongly suggest that ROCK2 gene polymorphisms may modify individual susceptibility to BD in the Turkish population.

Clinical significance of suppressor of cytokines signalling-3 mRNA expression from patients with non-Hodgkin lymphoma under chemotherapy

To date, little is known about blood immune marker changes that may be related to the development of Non Hodgkin Lymphoma (NHL) and treatment response with few serum biomarkers that could be useful in follow- up of the patients. To quantify the expression of suppressor of cytokine signalling-3-(SOCS-3) gene at the mRNA level in the peripheral blood of patients with NHL and correlate with clinical pathological features and response to treatment. Thirty patients with NHL and 20 healthy controls were enrolled in the study. The SOCS-3 mRNA level in peripheral blood (PB) was detected by semi-quantitative real-time polymerase chain reaction. Quantification of cytokines such as interleukin 6 and tumour necrosis factor alpha (IL-6 & TNF-α) were performed using sandwich enzyme-linked immunosorbent assays (ELISA). Increased expression of SOCS-3 mRNA in peripheral blood plus increased serum levels of IL-6 and TNF alpha from NHL cases with no complete remission after therapy. Higher levels of expression of SOCS-3 are associated with advanced disease, bone marrow involvement, extranodal involvement, poor performance status, B cell symptoms (fever, night sweats and weight loss) and high serum lactate dehydrogenase level which are evaluated by international prognostic index (IPI). Complete responses occur in 60% of patients with normal expression of SOCS-3 gene. Increased expression of SOCS-3 is common in diffuse large B cell lymphoma, CLL/small lymphocytic B cell lymphoma and follicular lymphoma. Over-expression of SOCS-3 mRNA from peripheral blood of NHL patients correlates with advanced disease and poor response to treatment. SOCS-3 mRNA expression in peripheral blood from NHL patients might be used to monitor response during treatment.

MAGE-4 gene m-RNA and TGF in blood as potential biochemical markers for HCC in HCV-infected patients

Progression from chronic hepatitis C virus infection to cirrhosis then to hepatocellular carcinoma usually results in some protein changes in peripheral blood. We evaluated MAGE-4 mRNA, TGFβ1 and AFP in peripheral blood as potential biochemical markers for diagnosis and prognosis of some complications of HCV infection. MAGE-4 mRNA in blood by reverse transcription polymerase chain reaction, serum TGF-Β1 and AFP by ELISA was assayed in seventy-five individuals who were classified into five groups: group I (control) comprised fifteen apparently healthy volunteers, group II involved fifteen HCV-infected patients without cirrhosis, group III involved fifteen HCV fifteen HCV-infected patients with cirrhosis, group IV included fifteen HCV-infected patients with cirrhosis and early stage HCC, and group V included fifteen HCV cirrhotic patients and late-stage HCC. We found that the frequency of positivity of MAGE-4 among the late hepatoma group was 40 %, while in the early hepatoma group the positivity was 6.7 %. The results for TGF-Β1 revealed a significant increase in serum TGF-Β1 in groups IV and V as compared to control, II, III groups. The obtained results of AFP showed a significant positive increase in serum AFP in groups IV and V when compared to groups II and III. Detection of MAGE-4 transcripts in blood, especially with follow-up survey, may help to predict the prognosis and monitoring of the response to the therapy, and serum TGF-Β1 level in HCC patients is directly correlated with metastasis and recurrence of tumors and increases gradually with the progression of HCC.

Abstract 4680: DNA dependent protein kinase as a new molecular target for treatment of non-small cell lung cancer

Abstract Prognosis with non-small cell lung cancer (NSCLC) has improved with the recent introduction of molecular targeted therapy. However, it is limited to patients with epidermal growth factor receptor (EGFR) mutations, who benefit from EGFR tyrosine kinase inhibitors (EGFR-TKI). A new molecular target is needed with other NSCLC patients. In general, chemotherapeutic agents cause DNA double-strand breaks (DNA DSB), leading to apoptosis of cancer cells; DNA repair reverses their effect, resulting in resistance to chemotherapy. DNA DSB repair can be mediated via two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). More than 90% of DSBs in mammalian cells are repaired by NHEJ, which is accomplished by core protein components including Ku70/Ku86, DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, Artemis, and Cernunnos-XLF. We recently reported that NK314, a dual inhibitor of topoisomerase IIα (Top2β) and DNA-PK, evidences a potent anti-tumor effect in adult T-cell leukemia-lymphoma (ATL). NK314 induced DNA DSBs and inhibited DNA repair mediated through degradation of the DNA-PK catalytic subunit (DNA-PKcs). In the present study, we examined the effect of NK314 in NSCLC cell lines and assessed DNA-PK as a possible molecular target for NSCLC. Twenty separate NSCLC cell lines were used, with IC50 varying from 46-193 nM. Levels of DNA-PKcs protein varied among the cell lines, whereas those of Ku70 and Ku86 did not. The inhibitory effect of cell growth was closely related to level of DNA-PKcs protein, but not to levels of other DNA-damage-activated protein kinases such as ATM or ATR. Establishment of predictive markers concomitantly with the development of a novel molecular target is indispensable for clinical application. Because detection of DNA-PKcs in peripheral blood is difficult, we investigated an alternative marker for predicting DNA-PKcs inhibitor. We previously found that heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), an RNA binding protein, interacts with DNA-PK, and we established a system for detecting hnRNP B1 mRNA in peripheral blood. We therefore examined the suitability of hnRNP B1 as an alternative biomarker for DNA-PK inhibitors. Close correlation between levels of DNA-PKcs and hnRNP B1 proteins was established using Western blot analysis with NSCLC cell lines (p=0.995). These results suggest that DNA-PK is a novel molecular target for NSCLC, and detection of hnRNP B1 might serve as an alternative molecular marker for the amount of DNA-PK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4680. doi:1538-7445.AM2012-4680

Paradoxical expression of IL-28B mRNA in peripheral blood in human T-cell leukemia virus Type-1 mono-infection and co-infection with hepatitis C Virus

BackgroundHuman T-cell leukemia virus type-1 (HTLV-1) carriers co-infected with and hepatitis C virus (HCV) have been known to be at higher risk of their related diseases than mono-infected individuals. The recent studies clarified that IL-28B polymorphism rs8099917 is associated with not only the HCV therapeutic response by IFN, but also innate immunity and antiviral activity. The aim of our research was to clarify study whether IL-28B gene polymorphism (rs8099917) is associated with HTLV-1/HCV co-infection.ResultsThe genotyping and viral-serological analysis for 340 individuals showed that IL-28B genotype distribution of rs8099917 SNP did not differ significantly by respective viral infection status. However, the IL-28B mRNA expression level was 3.8 fold higher in HTLV-1 mono-infection than HTLV-1/HCV co-infection. The high expression level was associated with TT (OR, 6.25), whiles the low expression was associated with co-infection of the two viruses (OR, 9.5). However, there was no association between down-regulation and ATL development (OR, 0.8).ConclusionHTLV-1 mono-infection up-regulates the expression of IL-28B transcripts in genotype-dependent manner, whiles HTLV-1/HCV co-infection down-regulates regardless of ATL development.

Diagnostic value of AFP, h-TERT and VEGF mRNAs in peripheral blood of patients with hepatocellular carcinoma

Diagnostic value of AFP, h-TERT and VEGF mRNAs in peripheral blood of patients with hepatocellular carcinoma

Correlation between expression of CK20 mRNA in peripheral blood and clinicopathological features and prognosis in patients with colorectal cancer

Correlation between expression of CK20 mRNA in peripheral blood and clinicopathological features and prognosis in patients with colorectal cancer

Peripheral blood mRNA expression patterns to differentiate hepatocellular carcinoma from other hepatic diseases

Peripheral blood genes expressions profiling (GeXP) have been convinced to be more specific for the diagnosis of cancer and other diseases, and the GeXP system provides an ideal method to analyze multiple genes expression in one normalized and equable system. We aim to differentiate hepatocellular carcinoma from other hepatic diseases based on peripheral blood and the GeXP system. Fifteen selected hepatic diseases related genes with two house-keeping genes for normalization were detected by the GeXP system. The diagnosis model was based on K nearest neighbor classifier and cross validation, and software based on MATLAB software was built for differential diagnosis of hepatic diseases. Eight hepatic related genes were demonstrated to show an obvious statistic difference in expressions while the K nearest neighbors classifier showed that the accuracy for normal controls, hepatitis B, liver cirrhosis, hepatocellular carcinoma and the Other group was separately 80.57 %, 78.17 %, 84.48 %, 73.24 % and 85.85 %. The set of validation has been carried out to assess the accuracy of Model Two and the accuracy was even higher than the set of building for the model, except for the hepatitis B (HBV) group. A sensitive and specific GeXP system of eight genes has been developed for the accurate differential diagnosis of hepatic disease.

Peripheral blood mRNA expression patterns to differentiate hepatocellular carcinoma from other hepatic diseases

Peripheral blood genes expressions profiling (GeXP) have been convinced to be more specific for the diagnosis of cancer and other diseases, and the GeXP system provides an ideal method to analyze multiple genes expression in one normalized and equable system. We aim to differentiate hepatocellular carcinoma from other hepatic diseases based on peripheral blood and the GeXP system. Fifteen selected hepatic diseases related genes with two house-keeping genes for normalization were detected by the GeXP system. The diagnosis model was based on K nearest neighbor classifier and cross validation, and software based on MATLAB software was built for differential diagnosis of hepatic diseases. Eight hepatic related genes were demonstrated to show an obvious statistic difference in expressions while the K nearest neighbors classifier showed that the accuracy for normal controls, hepatitis B, liver cirrhosis, hepatocellular carcinoma and the Other group was separately 80.57 %, 78.17 %, 84.48 %, 73.24 % and 85.85 %. The set of validation has been carried out to assess the accuracy of Model Two and the accuracy was even higher than the set of building for the model, except for the hepatitis B (HBV) group. A sensitive and specific GeXP system of eight genes has been developed for the accurate differential diagnosis of hepatic disease.

P5-01-16: The Detection of Circulating CK19 mRNA-Positive Cells in the Blood and the Mitotic Index of the Primary Tumor Have Independent Prognostic Value in Early Breast Cancer.

Abstract Background: Previous studies have shown that the molecular detection of CK-19 mRNA in peripheral blood and the mitotic index of primary tumor have prognostic value in early breast cancer. The aim of the present study was to assess the association between these variables. Patients and Methods: The primary tumors of 223 operable breast cancer patients (92 premenopausal and 131 postmenopausal) were evaluated for the mitotic activity index (MAI) classified either as &amp;lt;5/10, 6–10/10 and &amp;gt;10/10 or &amp;lt;10/10 and &amp;gt;10/10 mitoses/hpf using a standardized protocol as previously reported (Baak JP et al, Breast Cancer Res Treat 2009, 115:241–254). Peripheral blood was also collected before the start and after the end of adjuvant chemotherapy for detection of CK-19 mRNA-positive cells by RT-PCR as previously described (Stathopoulou et al, Clin Cancer Res 2003, 9:5145–5151). Results: After a median follow-up of 118 months, 75 (33.6%) patients have experienced disease relapse and 56 (25.1%) have died of breast cancer. MAI was strongly associated with disease-free survival (DFS) and overall survival (OS) (p&amp;lt;0.001 for both DFS and OS). Detection of CK19 mRNA-positive cells in the peripheral blood before but not after adjuvant chemotherapy was marginally associated with worse DFS (p=0.055) and OS (p=0.059). There was no correlation between MAI and CK19 mRNA detection before chemotherapy. Cox regression analysis revealed that both MAI and CK19 mRNA-positive cell detection before adjuvant chemotherapy were independent variables associated with decreased DFS (p&amp;lt;0.001 and p=0.038, respectively) and OS (p&amp;lt;0.001 and p=0.029, respectively). The interaction test showed no significant association between MAI and detection of CK19 mRNA-positive cells. Conclusion: MAI of the primary tumor and detection CK-19 mRNA-positive cells in the blood before the start of adjuvant chemotherapy in women with early breast cancer are two independent prognostic factors associated with clinical outcome. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-16.

Relationship of Th17 cells as well as interleukin (IL)-17A and IL-23R mRNA with psoriasis vulgaris

Objective To assess the number of peripheral blood Thl7 cells and mRNA expressions of IL-17A and IL-23R and their correlations with disease severity in patients with psoriasis vulgaris （PV）. Methods Tissue specimens were reseeted from the lesions of 25 patients with PV and normal skin of 10 human controls, and venous blood samples were obtained from 20 of the patients and all of the normal human controls. Reverse transcription PCR and flow cytometry were carried out to measure the mRNA levels of IL-17A and IL-23R in these tissue specimens and quantify the number of peripheral blood CD4＋IL-17＋ T lympbocytes. Psoriasis area and severity index （PASI） was calculated for these patients. Results A significant increase was observed in the IL-17A and IL-23R mRNA levels in the patients compared with the controls （0.996 ＋ 0.231 vs. 0.437 ~ 0.096, t-- 10.572, P 〈 0.05; 1.006 ~ 0.339 vs. 0.491 ~ 0.196, t = 6.015, P 〈 0.05）. The levels of both IL-17A mRNA and IL-23R mRNA were positively correlated with PASI （r~ = 0.67, 0.70, respectively, both P 〈 0.05）. The number of CD4＋IL-17＋ T lymphocytes in peripheral blood showed no significant differences between the patients and controls. No statistical correlation was observed between the counts of CD4＋IL-17＋ T lymphocytes and expression levels of IL-17A or 1L-23R mRNA in psoriatic lesions. Conclusions In patients with PV, there is an increase in the expressions of IL-17A and IL-23R mRNA in lesions, which are correlated with disease severity, while no significant change is observed in the number of peripheral blood CD4＊IL-17＋ T cells.

Expressional changes of neuregulin-1 gene mRNA in peripheral blood from schizophrenia patients

To explore the effect of anti-psychotic treatment on the expression of Neuregulin-1 (NRG1) mRNA in the peripheral blood lymphocytes of schizophrenia patients. The NRG1 mRNA in peripheral blood lymphocytes was measured using semi-quantitative reverse transcription (RT)-PCR in 80 first-onset schizophrenia patients, 37 sibling controls and 83 non-related controls. The patients were treated with risperdone and quetiapine for 4 weeks. Positive and negative symptom scale (PANSS) was used to evaluate the severity and clinical efficacy. Prior to the treatment, the expression of NRG1 mRNA expression was significantly lower in patients than other two groups (F=73.004, P=0.000). From the second week on, the level of NRG1 mRNA expression in patients became significantly higher than before and gradually increased, whilst no significant difference between sib and non-sib controls. Prior to the treatment, there was significant correlation (r=-0.232, P=0.038) between the level of NRG1 mRNA and PANSS scores. Four weeks after the treatment, a significant correlation between the reduction rate of PANSS and the change of NRG1 mRNA (r=0.27, P=0.016). The expression of NRG1 gene mRNA is associated with schizophrenia. Decreased expression of NRG1 may play a role in the development of schizophrenia, which can be improved by anti-psychotic drugs.

Indoleamine 2,3-Dioxygenase as a Predictor of Acute Rejection After Orthotopic Liver Transplantation in Rat Model

Background Indoleamine 2,3-dioxygenase (IDO) had been reported to correlate with immunomodulatory effects and the severity of acute rejection (AR) after liver transplantation. We sought to study the time course of IDO mRNA expression in peripheral blood to diagnose AR. Methods The rats were divided into 4 groups each consisting of 32 rats: group A, isograft Sprague-Dawley (SD)–SD); group B, acute rejection model (SD-Wistar); group C, cyclosporine (CsA)–induced acceptance model (SD-Wistar rats treated with CsA, and group D, short-term CsA-treated model. The peripheral blood and liver tissue samples were obtained on the day of operation as well as 1, 2, 3, 4, 5, 7, and 9 days thereafter. We performed reverse-transcription polymerase chain reaction, pathologic studies, and serum tests. Results Groups A and C showed low levels of IDO mRNA as well as normal values of aspartate transaminase (AST), total bilirubin (T-BIL), and alkaline phosphatase (ALP) without AR. Group B showed a dramatic rise in IDO mRNA on day 2 with increased AST, T-BIL, and ALP at day 4 and mild AR on day 5. Group D, showed dramatically up-regulated IDO mRNA on day 4 with significantly increased AST, T-BIL, and ALP day 5 and mild AR detected on day 7. The expression of IDO gene in peripheral blood tightly correlated with the severity of acute rejection ( P < .001). Conclusions Compared with the pathologic study detection of IDO mRNA in peripheral blood diagnosed AR in the rat model at an earlier stage.

The Role of Proteinase Inhibitor 9 (PI-9) and Granzyme B in Peripheral Leukocytes for GVL and GVHD After Allogeneic Hematopoietic Stem Cell Transplantation

Abstract 1984The serpin proteinase inhibitor 9 (PI-9) protects cells from serine protease granzyme B (GrB)- and perforin (PFR)-induced cytotoxicity and apoptosis by specifically inhibiting GrB. Graft-versus-leukemia (GVL) effect and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) are the reciprocal aspects of the established immunotherapeutic approach in hematopoietic malignancies, and are thought to be caused at least in part through GrB and PFR produced by donor-derived cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. However, GrB and PFR expressions with respect to a GVL effect, and the role of PI-9 in both GVHD and GVL, are unknown. In this study we analyzed the expression of GrB, PFR and PI-9 in acute leukemia patients to whom allogeneic HSCT was performed during remission. This study was performed under the approval of the ethical committee of Kobe University Graduate Schools of Medicine and Health Sciences, and all human samples were obtained from whom a written informed consent was obtained. We first investigated the age- and gender-related differences of expressions of PI-9 and GrB mRNA by quantitative RT-PCR in mononuclear and polymorphonuclear cells from healthy volunteers. GrB and PFR mRNAs were expressed almost exclusively in mononuclear cells that included CTLs and NK cells, while PI-9 was expressed both in mononuclear and polymorphonuclear cells. GrB and PFR expression levels were comparable among all age and gender groups. Intriguingly, however, the expression of PI-9 mRNA in lymphocytes gradually increased with age, and the PI-9 expression in the volunteer group aged 60 years old or older was significantly higher than the one in the group aged 20 to 39 years old (p=0.002). Meanwhile, the PI-9 expression in polymorphonuclear cells was comparable among all age groups, and there was no gender difference in PI-9 expression levels. Next, we analyzed the expressions of PI-9, GrB and PFR mRNAs in mononuclear and polymorphonuclear cells of 3 patients (male 2, female 1) with acute GVHD and 11 patients without acute GVHD (male 6, female 5). The GrB, PFR and PI-9 expression levels were comparable between patient groups with and without acute GVHD, both before transplantation and a week after engraftment. However, the GrB and PFR expressions prominently augmented when acute GVHD became overt in all 3 patients, while PI-9 expression level increased mildly. As a result, the ratio of GrB (or PFR) and PI-9 expressions was stable in patients with and without acute GVHD. Among them, 4 cohort patients eventually relapsed and 10 cohorts remained in remission. The GrB mRNA expression in lymphocytes one week after engraftment was 3-fold higher in 10 patients without relapse of primary disease than in 4 patients who eventually relapsed (P=0.044). This might suggest that GrB plays a role in eliciting a GVL effect as well as acute GVHD. On the other hand, PFR and PI-9 mRNA expressions were then relatively stable among patients both with and without later relapse (P=0.667; P=0.103), and the ratio of GrB (or PRF) and PI-9 expressions did not change. The expressions of GrB and PFR mRNAs in polymorphonuclear cells of all patients were under the detectable level throughout the course. Finally, we analyzed the expressions of GrB, PFR and PI-9 in leukocytes 6 months after HSCT, to see if these expressions might change in patients with chronic GVHD. Among 14 patients with chronic GVHD, the GrB expression increased (up to 6.2-fold) in 10 patients, but the PFR expression did not change. In contrast, the expression of PI-9 decreased profoundly in all of the cases both with and without chronic GVHD, relative to healthy volunteers (P=0.028). Importantly, the ratio of GrB and PI-9 expressions was significantly higher in patients with chronic GVHD than in those without GVHD (P = 0.035). In conclusion, (1) high PI-9 expression in elderly people might explain a mechanism of escape from tumor immunity in them, where PI-9 might inhibit functions of cytotoxic T cells and NK cells; (2) a high expression of GrB shortly after engraftment may be a novel biomarker for a GVL effect; and (3) a low level of PI-9 expression or an increased ratio of GrB/PI-9 later after allogeneic HSCT might be an important biomarker for cGVHD. Monitoring of both GrB and PI-9 mRNAs in peripheral blood after allogeneic HSCT could be useful for predicting the GVL effect, as well as for monitoring both acute and chronic GVHD. Disclosures:No relevant conflicts of interest to declare.

Iron Containing Compound Stimulates Expression of Pulmonary Hypertension Promoting Factor PlGF

Abstract 900We have previously reported elevated plasma levels of placental growth factor (PlGF) and endothelin-1 (ET-1) in patients with sickle cell disease with high estimated pulmonary artery systolic pressure, a marker of pulmonary hypertension, and gene transfer experiments in mice document that PlGF stimulates ET-1 expression and pulmonary hypertension. Among other markers, PlGF in SCD subjects is correlated with markers of excessive iron burden, including serum ferritin and transferrin, markers that have been previously associated with pulmonary hypertension in SCD in many studies. We therefore hypothesized that iron stimulates expression of PlGF. Because tissue postnatal expression of PlGF in vivo is restricted primarily to early erythroid cells, we chose K562 erythroleukemia cells as a cell culture model. We find that heme-bound iron (hemin) induces PlGF mRNA robustly in a dose-dependent and time-dependent fashion. The PlGF transcript rises robustly within hours of hemin stimulation, and reaches levels of 40 to 80-fold over baseline in a real time PCR assay. Heme analog compound, mesoporphyrin without iron fails to induce PlGF, but as a control induces hemeoxygenase-1 mRNA. Iron plus mesoporphyrin induces PlGF strongly, indicating a critical requirement for iron in inducing PlGF transcript. In promoter-reporter luciferase constructs transfected into K562 cells, hemin induces the human PlGF promoter significantly, and a minimal promoter construct contains binding sites for erythroid Kruppel-like factor (KLF-1). KLF-1 mRNA levels rise in hemin-stimulated K562 cells in parallel with PlGF, and in peripheral blood mRNA from SCD subjects and healthy controls, the level of KLF-1 transcript correlates closely with PlGF transcript (r=0.82, p<0.0001). Additional studies of KLF-1 are under way.Our results indicate for the first time a specific mechanistic pathway induced by iron that is linked in humans with SCD and in mice to vasculopathy and pulmonary hypertension. Rather a simple epiphenomenon of frequent transfusion or source of nonspecific oxidative stress, iron may trigger a KLF-1/PlGF/ET-1 linear mechanistic pathway to stimulate pulmonary hypertension. This suggests that PlGF levels are linked to the iron overload in patients with SCD. Further details in this pathway are under investigation. Disclosures:No relevant conflicts of interest to declare.

Th17/IL-17 Might Play a Protective Role in CLL Immunity

Abstract 2843 Background:Immune system disorders play an important role in the development and progression of CLL. Many deficiencies, contributing to disease progression, were found in T cell populations. Th17 cells represent a recently-discovered distinct lineage of helper T-cells, characterized by IL-17 secretion. IL-17 play an active role in inflammation and autoimmune diseases, however the role of IL-17 and Th17 cells in CLL immunopathogenesis remains undefined. We investigated IL-17A mRNA and IL-17A protein expression in peripheral blood (PB) CD4+ T cells and the plasma levels of IL-17 in 70 untreated patients with CLL in the context of clinical and immunological parameters. Th17 cell percentage was measured also in the bone marrow of CLL patients. The control group consisted of 20 healthy age-matched subjects. Materials and methods:Quantitative ‘real-time' reverse transcription-polymerase chain reaction (qRT-PCR) was used for the analysis of IL-17A mRNA in PB CD4+ T-cells. Intracellular IL-17A expression in CD3+/CD4+cells was analyzed using a three-color flow cytometry technique. Plasma levels of IL-17A, IL-4 and IL-10 were measured by ELISA. Results:None of the CD4+ T cells in the samples obtained from healthy volunteers (HV) contained detectable amounts of IL-17 mRNA, whereas it could be found in 5 out of 70 CLL samples. All CLL patients with detectable IL-17 mRNA in T-cells were in 0 Rai stage and negative both for ZAP-70 and CD38 expression. It is possible that IL-17 mRNA in these cells is expressed only for a short time thus evading detection by PCR, or that CLL cells contain low amounts of the IL-17 transcript and the RT-PCR method was not sufficiently sensitive to detect these small amounts. Frequently, in HV as well as in CLL patients, the percentage of CD4+ T cells with intracellular IL-17 expression in non-activation assays was lower than 1%, comparable to the level of autofluorescence. Despite the fact that IL-17 mRNA was not detected in the T cells of the majority of CLL patients, IL-17 protein was present in T cells after PMA and ionomycin stimulation. We found a significantly higher median percentage of CD4+/CD3+/IL-17+ cells in CLL patients (18.2%) than in HV (2.9%) (p<0.01). Median percentage of Th17 cells was significantly higher in CLL patients in stages 0–1 according to Rai, as compared with stages 2–4 (36.2% vs 4.5%, p<0.05). There were no differences in Th17 percentages between the PB and BM of CLL patients. Accordingly, the IL-17 plasma level was significantly higher in CLL patients than in HV (101.9 pg/ml vs 75.6 pg/ml; p<0.05) and there was a significant correlation between Th17 cell percentage and IL-17 plasma level. There was also significant correlation between the percentages of Th17 and iNKT cells (R=0.56; p<0.05), and inverse correlations between the percentage of Th17 and: Treg cells (R=−0.28; p<0.05), CD4+IL-4+ (R=−0.55; p<0.05) and CD4+TNF+ T cells (R=−0.59; p<0.01). The percentage of Th17 inversely correlated with β2-microglobulin serum level (R=−0.29; p<0.05) and the expression of ZAP-70 (R=−0.244; p<0.05) and CD38 (R=−0.33; p<0.01). The plasma level of IL-17 inversely correlated with the stage of disease (R=−0.23; p<0.05), IL-10 (R=−0.56; p<0.01) and TNF (R=−0.66; p<0.01) plasma levels, CD38 antigen (R=−0.25; p<0.05) and ZAP-70 expression (R=−0.28; p<0.05). In patients requiring therapy during the observation period, the median percentage of Th17 cells and median plasma IL-17 level were significantly lower comparing to untreated ones (5.7% vs 23.1%, p<0.05; 53.8 vs 95.6 pg/ml, p<0.05; respectively). Moreover, median plasma IL-17A level correlated with the time from CLL diagnosis to the start of therapy (R=0.49; p<0.01). Conclusions: both the Th17 cell percentage, and the IL-17 plasma level are significantly higher in the PB of untreated CLL patients compared with the HV, and IL-17 mRNA expression was found only in the T cell of CLL patients, but not in the control group. Correlations found with disease activity parameters, the subsets of T cells involved in tumor surveillance, and also with the length of time from diagnosis to the start of therapy, suggest an important protective role for Th17 cells in CLL immunity. Disclosures:No relevant conflicts of interest to declare.