Expression In Peripheral Blood Cells Research Articles

Expression of DOG1 in peripheral blood cells of patients with gastrointestinal stromal tumor

Background and study aimsThe diagnosis and surveillance of gastrointestinal stromal tumor (GIST) rely on pathology and immunochemistry (IHC), making it complicated and invasive. Noninvasive and convenient biomarkers of this disease need to be explored. The high specificity and sensitivity of IHC in detecting GIST 1 (DOG1) in biopsy indicate that it is also expressed in circulating tumor cells of the blood and may be an ideal biomarker for GIST. This aimed to detect the expression of DOG1 in peripheral blood cells (PBCs) and determine the relationship between DOG1 expression and clinical factors. Patients and methodsA total of 45 patients with GIST and 46 healthy controls (HCs) were recruited from the Second Affiliated Hospital of Nanchang University between December 2015 and June 2018. PBCs were isolated from peripheral venous blood by density gradient centrifugation. RNA was extracted from PBCs, and DOG1 mRNA was detected by quantitative reverse transcription polymerase chain reaction. DOG1 mRNA expression between GIST and HC was compared, and the relationship between clinical factors and DOG1 was also analyzed. ResultsDOG1 mRNA expression in PBCs was significantly higher in patients with GIST than that in HCs (3.326 [1.942–5.328] versus 0.744 [0.269–1.087], p < 0.01). The specificity and sensitivity were 88.9% and 89.1%, respectively (AUC = 0.912). Tumor diameter and risk of aggressive behavior were correlated with DOG1 expression, and other clinical factors (sex, age, location, number of phase-splitting cells, Ki-67 index, metastatic status) did not show any relationship with DOG1 expression. However, clinical factors, including tumor diameter and risk grade, were not independent factors in DOG1 expression when multivariate analysis was conducted. ConclusionDOG1 expressions were significantly higher in patients with GIST than that in HCs. Tumor diameter and risk classification correlated with DOG1 expression but were not independent factors. DOG1 in PBCs is a promising noninvasive biomarker for GIST.

MO440KLOTHO AS A BIOMARKER OF SUBCLINICAL CARDIOVASCULAR DISEASE IN CHRONIC KIDNEY DISEASE: A PROOF-OF-CONCEPT STUDY*

Abstract Background and Aims Cardiovascular disease (CVD) is the major cause of mortality among chronic kidney disease (CKD) patients. Reductions in serum Klotho levels are related to the prevalence of CVD in CKD patients. However, it is unclear whether circulating Klotho, and its expression in peripheral blood cells (PBCs), are associated with subclinical atherosclerotic cardiovascular disease (sCVD) in these subjects. In this proof-of-concept study, we analyzed in a group of CKD patients the relationship between Klotho and two markers of sCVD: ankle-brachial index (ABI) and carotid intima-media thickness (CIMT). Method Gene expression in PBCs and serum levels of Klotho and inflammatory cytokines (TNF, IL6 and IL10) were measured in 103 CKD patients (stages 3-4), older than 18 years of age, and without known atherosclerotic cardiovascular disease. Biochemical data were obtained following standardized clinical methods. The presence of sCVD was defined as ABI &amp;lt; 0.9 and/or CIMT ≥ 0.9 mm. Patients with ABI values ≥ 1.3 were excluded. Results Patients with sCVD presented lower serum and PBCs expression levels of Klotho (P&amp;lt;0.001 for both). Stratified analysis showed that upper tertiles of both serum and PBCs expression levels of Klotho presented significantly higher ABI (P&amp;lt;0.001 for both Klotho determinations) and lower CIMT (P&amp;lt;0.001 for serum levels and P&amp;lt;0.01 for KL expression in PBCs), which resulted in a lower prevalence of sCVD (P&amp;lt;0.001 for both determinations of Klotho). Correlation analysis showed that both serum and PBCs mRNA Klotho levels were positively correlated with ABI (r=0.556, P&amp;lt;0.0001; and r=0.373, P&amp;lt;0.0001, respectively) and inversely correlated with CIMT (r=-0.541, P&amp;lt;0.0001; and r=-0.437, P&amp;lt;0.0001, respectively). Among inflammatory markers, only serum IL6 levels presented significant associations with sCVD, being inversely related with ABI (r=-0.568, P&amp;lt;0.0001) and positively associated with CIMT (r=0.558, P&amp;lt;0.0001). Multiple regression analysis with ABI and CIMT as dependent variables demonstrated that both Klotho variables, together with serum IL6, were positively and significantly associated with ABI (adjusted R2=0.511, P&amp;lt;0,0001) and CIMT (adjusted R2=0.445, P&amp;lt;0,0001) values, independently of traditional and emergent cardiovascular risk factors. Multivariate logistic regression, using the presence/absence of sCVD as the dependent variable, showed that circulating Klotho, and its expression in PBCs constituted independent protective factors for sCVD [OR (95% CI): 0.993 (P=0.002) and 0.231 (P=0.025), respectively]. Receiver operating curve (ROC) analysis pointed to the prognostic ability for sCVD of serum Klotho (area under the curve [AUC]: 0.817, 95% CI: 0.736–0.898, P&amp;lt;0.001) and its gene expression in PBCs (AUC: 0.742, 95% CI: 0.647–0.836, P&amp;lt;0.001). Conclusion The reductions in serum soluble and PBCs expression levels of Klotho in CKD patients are independently associated with the presence of sCVD. Further research exploring whether therapeutic approaches to maintain or elevate the Klotho level could reduce the impact of CVD in CKD patients is warranted.

FOXO1 and ANGPT2 relative gene expression in non-ST-segment elevation myocardial infarction among patients with or without type 2 diabetes.

IntroductionIt is well known that chronic hyperglycemia or chronic inflammation leads to both FOXO1 and Ang-2 gene (ANGPT2) expression induction in endothelial cells. ANGPT2 and FOXO1 relative gene expression in peripheral blood cells in diabetes and myocardial ischemia were not researched extensively.AimOur objective was to evaluate ANGPT2 and FOXO1 gene expression in peripheral blood cells in patients with non-ST elevation myocardial infarction (NSTEMI), both with and without type 2 diabetes mellitus (T2DM), and compare them to the results obtained from T2DM and control subjects.Material and methodsThis was a multi-center, prospective study of 138 NSTEMI patients with/without T2DM, T2DM and a control group. FOXO1, ANGPT2, TBP (TATA box binding protein – as a reference gene) gene expression levels in peripheral blood cells were measured in each patient. Electrocardiography and echocardiography with assessment of ejection fraction (EF) were performed. Patients with NSTEMI underwent urgent (< 24 h) coronarography and the SYNTAX score and GRACE 2.0 score were calculated.ResultsThe ANGPT2 gene relative expression in buffy coat in the analyzed samples was very low and detectable only in 11 patients from all groups (8.66% of all patients). The level of FOXO1 gene relative expression was significantly higher in patients with NSTEMI (median relative expression = 1.39) than in non-NSTEMI patients (median = 1.09) (W = 1578, p < 0.05) regardless of the presence of T2DM. The FOXO1 gene relative expression was not correlated with GRACE 2.0 score or SYNTAX score of NSTEMI patients. We did not observe any significant change in FOXO1 gene expression after successful angioplasty.ConclusionsOn the basis of our results we can conclude that analyzing the ANGPT2 gene relative expression in peripheral blood cells has no role in assessment of CAD complexity among patients with and without T2DM. FOXO1 gene relative expression in blood peripheral cells is elevated in patients with NSTEMI regardless of the presence of T2DM. FOXO1 expression does not decrease after successful percutaneous coronary intervention and is not correlated with the severity of CAD in patients with NSTEMI.

Circadian rhythms of melatonin, cortisol, and clock gene expression in the hyperacute phase of wake-up stroke: study design and measurement.

Circadian rhythms of melatonin, cortisol, and clock gene expression in the hyperacute phase of wake-up stroke: study design and measurement.

Determination of PD-1 expression in peripheral blood cells in patients with endometriosis

In patients with endometriosis, ectopic endometrial tissues can escape from immune system control and survive in other tissues. The pathophysiology of endometriosis is still not fully understood. In this study, we aimed to clarify the pathophysiology of endometriosis, which is thought to be a benign but infiltrative cancer type, which has many similarities with cancer biology by determining PD-1 expression in patients with endometriosis. In this study, n = 73 cases who underwent surgery or examination at the Obstetrics and Gynecology Clinic of Sivas Cumhuriyet University Faculty of Medicine and diagnosed as endometriosis in the biopsy material taken with the pre-diagnosis of endometriosis constituted the patient group. The control group consisted of n = 64 healthy subjects without concomitant malignancy or chronic inflammatory disease. Venous whole blood samples were obtained from the study groups. PD-1 and PD-L1 levels were determined by the ELISA method from serum and plasma samples. PD-1 gene expression level was determined by RT-PCR. The PD-1 level was found to be approximately 350 ± 150 ng/L and 45 ± 17 ng/L in endometriosis and control group, respectively. While the PD-L1 level was approximately 760 ± 108 ng/L in the patients, this level was 140 ± 14 ng/L in the controls. According to the RT-PCR results, the expression of the PD-1 gene 10 times higher compared to the controls. Conclusion: The identified increase of PD-1 levels and gene expression in endometriosis groups show that immunotherapy may be used in the treatment of endometriosis.

Toll-like receptor 2/4 inhibitors can reduce preterm birth in mice.

ObjectivesPreterm birth (PTB) occurs in 5% to 18% of newborns. However, the underlying inflammatory mechanisms have not been elucidated.MethodsWe established a mouse model of infection-associated PTB. Physical signs in pregnant mice with or without lipopolysaccharide (LPS) treatment were observed, and the frequencies of Toll-like receptor (TLR)2- and TLR4-positive CD11b+ cells were analyzed. Cytokine levels in plasma and pathological changes were assessed following LPS treatment. A rescue experiment was used to probe potential immunologic mechanisms underlying PTB.ResultsLymphocyte infiltration could be observed in the placentas of mice following intrauterine injection with LPS. The percentage of inflammatory cells decreased 12 hours after treatment. Moreover, TLR2 and TLR4 expression in peripheral blood cells was significantly increased 4 hours after intraperitoneal injection of LPS. Peak TLR2 and TLR4 expression in peripheral blood cells occurred 8 hours post-treatment. TLR4 and TLR-2/4 inhibitors reduced levels of interleukin-10, interferon-γ, and tumor necrosis factor-α in peripheral blood and delayed PTB.ConclusionsTLR2 and TLR4 inhibition could play important roles in PTB.

The Relationship between the Expression of Fatty Acyl Desaturase 2 (fads2) Gene in Peripheral Blood Cells (PBCs) and Liver in Gilthead Seabream, Sparus aurata Broodstock Fed a Low n-3 LC-PUFA Diet.

The principle aim of this study is to elucidate the relationship between the fatty acid desaturase 2 gene (fads2) expression pattern in peripheral blood cells (PBCs) and liver of gilthead seabream (GSB), Sparus aurata broodstock in order to determine the possible use of fads2 expression as a potential biomarker for the selection of broodstock. This selection could be utilized for breeding programs aiming to improve reproduction, health, and nutritional status. Passive Integrated Transponder (PIT)-tagged GSB broodstock (Male-1.22 ± 0.20 kg; 44.8 ± 2 cm and female-2.36 ± 0.64 kg; 55.1 cm) were fed a diet containing low levels of fish meal and fish oil (EPA 2.5; DHA 1.7 and n-3 LC-PUFA 4.6% TFA) for one month. After the feeding period, fads2 expression in PBCs and liver of both male and female broodstock were highly significantly correlated (r = 0.89; p < 0.001). Additionally, in male broodstock, liver fads2 expression was significantly correlated (p < 0.05) to liver contents in 16:0 (r = 0.95; p = 0.04) and total saturates (r = 0.97; p = 0.03) as well as to 20:3n–6/20:2n–6 (r = 0.98; p = 0.02) a Fads2 product/precursor ratio. Overall, we found a positive and significant correlation between fads2 expression levels in the PBCs and liver of GSB broodstock. PBCs fads2 expression levels indicate a strong potential for utilization as a non-invasive method to select animals having increased fatty acid bioconversion capability, better able to deal with diets free of fish meal and fish oil.

Gene expression in peripheral blood cells for differentiation of active and latent tuberculosis infection in children and adolescents

The objective: to analyze the expression of certain genes in the blood cells of children and adolescence to differentiate the active and latent phases of tuberculosis infection.Subjects and methods. Peripheral blood samples collected in 36 pediatric patients with latent tuberculosis infection and 24 patients aged 1 to 16 years undergoing in-patient treatment for pulmonary tuberculosis were tested. A modified method for isolating messenger RNA and reverse transcriptional polymerase chain reaction was used to identify the transcription of six genes selected for analysis.Results. In a comparative study of the expression values of six promising genes in blood cells in the study of two groups of children and adolescents with latent and active tuberculosis infection, it was found that the most differentiating feature for determining active tuberculosis infection was a significantly higher level of expression of PDCD1 gene encoding PD1 lymphocyte receptor. At the same time, the sensitivity to detect the active infection was found to be 95.8%, specificity – 94.4%, the accuracy of the positive prognosis of active tuberculosis infection was 93.3%.

Abstract DP-003: TOWARD A HEMATOPOIETIC STEM CELL-BASED PROPHYLACTIC IMMUNO-GENE THERAPY APPROACH FOR OVARIAN CANCER

Abstract Our ultimate goal is to develop a long-lasting, cost-efficient, and technically simple approach that allows for the immuno-prophylaxis of cancer in patients with high-risk for disease recurrence and, ultimately, in patients with cancer-predisposing inherited mutations. Our approach is based on in vivo genetic modification of hematopoietic stem cells (HSCs). Because HSCs are self-renewing and give rise to all blood cell lineages, they provide a life-long source of transgene modified myeloid and lymphoid cells that, during tumor development, infiltrate the tumor and support tumor growth. Our in vivo HSC transduction approach involves subcutaneous injections of G-CSF/AMD3100 to mobilize HSCs from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating, helper-dependent adenovirus (HD-Ad5/35++) vector system. In preliminary studies we used an integrating HDAd5/35++ vector expressing GFP and mgtmP140K under the ubiquitously active EF1a promoter. We demonstrated in adequate mouse models that HSCs transduced in the periphery home back to the bone marrow where they persist long-term. Transgene expression in peripheral blood cells can be achieved by O6BG/BCNU injection. Short-term exposure of in vivo transduced mice to low O6BG/BCNU doses resulted in stable GFP expression in 80% of peripheral blood cells and, in mice with implanted syngeneic tumors, in 80% of tumor infiltrating leukocytes. The predominant GFP-positive cell type in two syngeneic tumor models was tumor-associated neutrophils (TANs) (Ly6G+) and macrophages (TAMs) (F4/80+/MHCII+). GFP+ TANs and TAMs were detectable at very early stage of tumor development. To avoid adverse reactions using our in vivo HSC transduction/selection approach, the expression of therapeutic transgenes has to i) be localized to the tumor, ii) be automatically activated only when the tumor begins to develop, and iii) cease when the tumor disappears. To develop such an expression system, we determined (by miRNA-Seq and miRNA-array) the micro-RNA profile in GFP+ cells isolated from the bone marrow HSCs, spleen, PBMCs, and tumors of in vivo transduced mice. We found four miRNAs that were expressed at high levels in HSCs, splenocytes and PBMCs, but were absent in tumor-associated leukocytes. By inserting four copies of the corresponding target sites into the 3'UTR of our GFP transgene, the corresponding mRNA is degraded in all cells except tumor-associated leukocytes allowing for tumor-restricted transgene expression. Studies on validating this system using GFP as a reporter gene are nearly completed. Furthermore, we have generated HDAd5/35++ vectors expressing scFv specific to mouse CTLA4 or PDL1 under miRNA control. We have performed in vivo HSC transduction-O6BG/BCNU treatment with these vectors and will implant syngeneic tumors at the beginning of August. We expect that intratumoral anti-CTLA4 or anti-PDL1 expression from TANs/TAMs at early stages of tumor development will overcome the immunosuppressive tumor environment allowing effector T-cells to stop tumor growth. The outcome of these studies will be presented. We have also started testing the prophylactic efficacy of our approach in oncogene-transgenic mice that develop spontaneous tumors. Citation Format: Chang Li, Paul Valdmanis, Andre Lieber. TOWARD A HEMATOPOIETIC STEM CELL-BASED PROPHYLACTIC IMMUNO-GENE THERAPY APPROACH FOR OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr DP-003.

Expression of LRP1 and CHOP genes associated with peripheral neuropathy in type 2 diabetes mellitus: Correlations with nerve conduction studies

AimDiabetic peripheral neuropathy (DPN) is a frequent and debilitating complication of diabetes mellitus. The low-density lipoprotein receptor-related protein-1 (LRP-1) is a multifunctional cell surface receptor playing critical roles in lipoprotein metabolism and several cell signaling processes. C/EBP homologous protein (CHOP) is a main conduit to endoplasmic reticulum stress-induced apoptosis. We aimed to investigate LRP1 and CHOP gene expression in peripheral blood cells of type 2 diabetes mellitus (T2DM) subjects to clarify its possible relation to DPN pathogenesis. MethodThe study included 20 non-complicated T2DM subjects, 20 subjects with DPN and 20 healthy controls. Quantitative real time PCR was used to study gene expression. ResultsThere was a significant reduction in LRP1 mRNA expression and a significant increase in CHOP mRNA expression in subjects with DPN compared to non-complicated group and healthy controls. Both LRP1 and CHOP expression levels were inversely correlated, and both showed significant correlation with HbA1c, hyperlipidemia, hs-CRP, and different electrophysiological parameters. Receiver operating characteristics (ROC) analysis suggested that both LRP1 and CHOP mRNA expression and hs-CRP levels had great potential advantages to predict the progression of DPN. ConclusionLRP1 and CHOP might be involved in DPN pathogenesis and progression, thus providing opportunities for early detection and treatment.

Effects of Abnormal Iron Metabolism on EPO-STAT5 Signaling Pathway in Anemia Patients

To study the effects of iron metabolism abnormality on EPO-STAT5 signaling pathway in anemia patients. According to diseases, the patients were divided into 3 groups: lower risk myelodysplastic syndrome (MDS) group (30 cases) including 14 cases of non-iron over load and 16 cases of iron over load, 12 cases of them were treated by iron chelation therapy; anemia of chronic disease (ACD) group (12 cases) and iron deficiency anemia (IDA) group (12 cases). In addition, the healthy control group was selected. The iron metaloslism index (SF, SI, TIBC), serum level of EPO, plasma level of P-STAT5 and STAT-5 mRNA expression in peripheral blood cells were detected and compared in different groups. Moreover, the effects of iron metabolism abnormality on the expression of EPO and STAT5 in anemia patients were analyzed. compared with non-iron over load group, the EPO level in iron over load group significantly increased (P<0.05), the expression of STAT5 mRNA and P-STAT5 significantly decreased (P<0.05). After iron chelation therapy, the EPO level in serum significantly decreased (P<0.05), the expression of STAT5 mRNA and P-STAT5 was up-regulated significantly (P<0.05). Compared with healthy control group, the expression of EPO in ACD group was down-regulated significantly, while the expression of STAT5 mRNA was not different, but the P-STAT5 expression was down-regulated significantly (P<0.05). Compared with the healtly control group, the EPO expression in IDA group was enhanced significantly (P<0.05), the expression of STAT5 mRNA and P-STAT5 were also significantly enhanced (P<0.05). The excessive iron load or chronic inflammation may inhibit the activation of EPO-STAT5 signaling pathway and aggravate the anemia.

Increased PD-1 MRNA Expression in Peripheral Blood Cells of ER+ and PR+ Breast Cancer Patients and Its Unfavorable Prognostic Value

Background: The clinical significance of programmed cell death 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) expression on tumor infiltrating lymphocytes in breast cancer patients has been confirmed, while that of peripheral blood cells derived immune molecules remain unclear. Aim: We aimed to investigate the clinical importance of immune molecules, including PD-1 and CTLA-4, expression in peripheral blood cells of breast cancer patients, especially in terms of the relationship between immune molecules and estrogen receptor (ER)/progesterone receptor (PR) status, as well as their prognostic values. Methods: We enrolled 109 breast cancer patients, including 52 cases before surgery and neoadjuvant treatment (PreS group), 18 cases postsurgery and adjuvant chemoradiotherapy (PostS group), 39 metastatic cases presalvage treatment (Met group), and 21 age- and sex-matched healthy volunteers). The mRNA abundance of PD-1, CTLA-4, IL-2 receptor alpha (IL-2Rα), and cluster of differentiation 28 (CD28), forkhead box P3 (FOXP3), transforming growth factor beta (TGF-β), and interleukin-10 (IL-10) in pretreatment peripheral blood were analyzed by quantitative real-time PCR. Results: ER+ breast cancer patients showed significant higher mRNA levels of PD-1, CTLA-4, IL-2Rα, and CD28 with fold changes of 10.8, 2.4, 5.0, and 3.8, respectively ( P &lt; 0.05) than that of ER− cases. Similarly, PR+ patients showed increased levels of PD-1, CTLA-4, and CD28 with fold changes of 6.7, 2.0, and 2.5, respectively ( P &lt; 0.05) comparing to that of PR− cases. Patients in PreS group and Met group showed higher mRNA levels of PD-1, CTLA-4, IL-2Rα, CD28, FOXP3, TGF-β, and IL-10 than PostS group and healthy volunteers. Univariable analysis revealed that high PD-1 expression was associated with poorer progression-free survival (PFS) in metastatic breast cancer patients (5.9 vs 14.6 months, HR: 2.47, 95% CI: 1.22-5.02, P = 0.046). Meanwhile, the prognostic value of PD-1 was remained in multivariate analyses (HR: 2.22, 95% CI: 1.04-4.73, P = 0.039). Conclusion: Increased PD-1, CTLA-4, and CD28 mRNA abundance were showed in breast cancer patients and ER+/PR+ cases, which may provide the rationale for combining checkpoint inhibitors with endocrine therapy for breast cancer treatment. Furthermore, PD-1 is a promising prognostic biomarker for metastatic breast cancer.

Alteration of microRNA 340-5p and Arginase-1 Expression in Peripheral Blood Cells during Acute Ischemic Stroke.

Acute stroke alters the systemic immune response as can be observed in peripheral blood; however, the molecular mechanism by which microRNA (miRNA) regulates target gene expression in response to acute stroke is unknown. We performed a miRNA microarray on the peripheral blood of 10 patients with acute ischemic stroke and 11 control subjects. Selected miRNAs were quantified using a TaqMan assay. After searching for putative targets from the selected miRNAs using bioinformatic analysis, functional studies including binding capacity and protein expression of the targets of the selected miRNAs were performed. The results reveal a total of 30 miRNAs that were differentially expressed (16 miRNAs were upregulated and 14 miRNAs were downregulated) during the acute phase of stroke. Using prediction analysis, we found that miR-340-5p was predicted to bind to the 3'-untranslated region of the arginase-1 (ARG1) gene; a luciferase reporter assay confirmed the binding of miR-340-5p to ARG1. miR-340-5p was downregulated whereas ARG1 mRNA was upregulated in peripheral blood in patients experiencing acute stroke. Overexpression of miR-340-5p in human neutrophil and mouse macrophage cell lines induced downregulation of the ARG1 protein. Transfection with miR-340-5p increased nitric oxide production after LPS treatment in a mouse macrophage cell line. Our results suggest that several miRNAs are dynamically altered in the peripheral blood during the acute phase of ischemic stroke, including miR-340-5p. Acute stroke induces the downregulation of miR-340-5p, which subsequently upregulates ARG1 protein expression.

Peripheral-blood gene expression profiling studies for coronary artery disease and its severity in Xinjiang population in China

BackgroundAlterations in gene expression in peripheral blood cells play a curtail role in the presence and extent of coronary artery disease (CAD), but its severity reflected by gene expression alterations in peripheral blood cells is still unknown in Xinjiang population in China.MethodsGlobal gene expression profiling in peripheral blood was used to explore differentially expressed genes in coronary artery stenosis patients. RNA was extracted from peripheral blood of 9 controls without coronary stenosis and 21 cases with angiographically CAD. The extent of CAD severity was categorized angiographically as no CAD, mild CAD (20 to 50% luminal diameter stenosis [LDS]), moderate CAD (50 to 75% LDS) and severe CAD (≥75% LDS). Differentially expressed genes related with CAD severity from peripheral blood cells were screened by linear mixed effects analysis using the lme4 package in R. Then the differentially expressed genes that gradually up-regulated or down-regulated were enriched by Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.ResultsThe most significantly enrichments were toll-like receptor signaling pathway, immune responses, translational processes, cellular growth, inflammation and metabolic processes. Combined with NCBI-GeneRIF and PubMed analysis, we focused on the 12 genes associated with toll-like receptor signaling pathway in the extent of coronary artery stenosis patients. Receiver operating characteristic (ROC) analysis of 12 genes associated with toll-receptor signaling pathway in the 236 CAD patients from GEO database demonstrated that 12 genes expression could predict severe CAD with an area under the curve of 0.67, sensitivity of 77.65% and specificity of 51.52%.ConclusionThese results suggest that 12 genes associated with toll-like receptor signaling pathway in peripheral-blood cells reflect the presence and extent of CAD severity in Xinjiang population in China.

Cocaine-induced epigenetic DNA modification in mouse addiction-specific and non-specific tissues

Cocaine-related DNA methylation studies have primarily focused on the specific brain regions associated with drug addiction (e.g., the nucleus accumbens, NAc). To date, no studies have focused on the complex role of both DNA methylation and demethylation in the mechanisms of psychostimulant-induced addiction in the brain and peripheral tissues. Therefore, in this study, we evaluated cocaine treatment and withdrawal (animals were withdrawn from seven days of repeated injections of cocaine that caused behavioral sensitization) effects on epigenetic DNA modifiers (i.e., DNA methyltransferases, [DNMTs] and ten-eleven translocation enzymes [TETs]) in an addiction-specific brain region (NAc), a structure outside the mesolimbic dopaminergic system (cerebellum, Cer), and in peripheral blood cells (PBCs). Using a mouse behavioral sensitization model, we demonstrated that acute cocaine (AC; 0.5 h) treatment significantly decreased Dnmt1, Dnmt3a, Tet1, and Tet2 mRNA levels in the NAc and PBC, whereas at 24 h after AC treatment, Dnmt mRNA expression and enzyme activity levels were significantly increased. Acute procaine treatment caused the opposite effect on the Dnmt3a mRNA level in PBCs; this outcome suggests that the inhibition of voltage-gated sodium channels may be the mechanism that alters Dnmt expression in PBCs. Cocaine withdrawal is associated with increased expression of Dnmts in the NAc, Cer and PBCs and the decreased expression of Tet1 and Tet3 in the NAc. Additionally, cocaine withdrawal increased DNMT but decreased TET activity levels, and these changes were associated with enhanced global and selected candidate gene promoter-region DNA methylation and hydroxymethylation levels in the NAc and PBCs. Together, these data indicate that cocaine treatment and withdrawal affect the expression of epigenetic DNA modifiers in both addiction-specific brain structures and structures outside of the mesolimbic dopaminergic system and PBCs.

CD26 mRNA expression in Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a complex autoimmune disease which involves several organs.CD26 is a multifunctional molecule that has an extracellular domain with dipeptidyl peptidase IV activity which digests crucial inflammatory molecules. CD26 plays an important role in T-cell activation and enhances immune responses. This study was carried out to evaluate the level of CD26 gene expression in SLE patients. Forty-six SLE patients and 44 healthy controls voluntarily participated in this study. Based on the SLE disease activity index (SLEDAI), the patients were divided into two subgroups, those with active disease (n=24) and those with inactive disease (n=22). Patients were also subgrouped according to renal involvement, as those with lupus nephritis (n=17) and those without lupus nephritis (n=29). Their CD26 mRNA levels in peripheral blood cells were analyzed by quantitative RT-PCR. CD26 mRNA expression increased 3.6-fold in SLE patients in comparison with the controls (p 0.05). Although CD26 mRNA expression in patients with lupus nephritis was 2.76-fold higher than those without nephritis, the difference was not statistically significant (p>0.05). CD26 gene expression in peripheral blood cells of SLE patients significantly increased over that of the controls. This increase was not affected by the disease activity nor did it show any significant correlation with complications in organs.

Identification of disulfiram as a secretase-modulating compound with beneficial effects on Alzheimer\u2019s disease hallmarks

ADAM10 is a metalloproteinase acting on the amyloid precursor protein (APP) as an alpha-secretase in neurons. Its enzymatic activity results in secretion of a neuroprotective APP cleavage product (sAPP-alpha) and prevents formation of the amyloidogenic A-beta peptides, major hallmarks of Alzheimer’s disease (AD). Elevated ADAM10 levels appeared to contribute to attenuation of A-beta-plaque formation and learning and memory deficits in AD mouse models. Therefore, it has been assumed that ADAM10 might represent a valuable target in AD therapy. Here we screened a FDA-approved drug library and identified disulfiram as a novel ADAM10 gene expression enhancer. Disulfiram increased ADAM10 production as well as sAPP-alpha in SH-SY5Y human neuronal cells and additionally prevented A-beta aggregation in an in vitro assay in a dose-dependent fashion. In addition, acute disulfiram treatment of Alzheimer model mice induced ADAM10 expression in peripheral blood cells, reduced plaque-burden in the dentate gyrus and ameliorated behavioral deficits. Alcohol-dependent patients are subjected to disulfiram-treatment to discourage alcohol-consumption. In such patients, enhancement of ADAM10 by disulfiram-treatment was demonstrated in peripheral blood cells. Our data suggest that disulfiram could be repurposed as an ADAM10 enhancer and AD therapeutic. However, efficacy and safety has to be analyzed in Alzheimer patients in the future.

Abstract B001: A phase 1 trial of RGX-104, a first-in-class immunotherapy targeting the liver-X nuclear hormone receptor (LXR), in patients with refractory malignancies

Abstract Background: RGX-104 is a small-molecule LXR agonist that activates the expression of the ApoE gene. We have identified the LXR/ApoE pathway as a novel innate-immune checkpoint that stimulates antitumor immunity. LXR agonist-mediated ApoE induction depletes circulating and tumor-infiltrating myeloid derived suppressor cells (MDSCs), resulting in activation of cytotoxic lymphocytes (CTLs) in murine tumor models. Treatment with RGX-104 as monotherapy elicited significant antitumor activity in a variety of preclinical models, including B16F10, LLC, and CT26. Additionally, combining RGX-104 with a PD-1 antagonist yielded synergistic antitumor activity in PD-1 resistant models. These data motivated studies of RGX-104 in patients with refractory cancer. Methods: We are conducting a first-in-human, dose escalation and expansion phase 1 study of RGX-104 in patients with refractory solid tumors or lymphomas. Safety, PK, PD, efficacy, and immunologic activity are being assessed. Results: RGX-104 was dosed orally for 21 days with a 1-week rest (28 day cycle), at 120 mg QD (n=3) and 240 mg QD (n=4). Tumor types in cohorts 1 and 2 included colon, uterine, renal, melanoma, cholangiocarcinoma, and Ewing’s sarcoma. PK analysis demonstrated dose-proportional drug increases in AUC and a t1/2 of ~7 hours with oral administration. Induction of ApoE gene expression in peripheral blood cells (median 3.57-fold; mean 11.61-fold at steady-state) correlated with RGX-104 drug exposure, indicating robust LXR target engagement. Monitoring of relevant immune-cell populations in peripheral blood revealed substantial MDSC depletion in 6/6 evaluable patients, with up to 95% depletion of granulocytic (CD33+CD15+HLA-DR-/low; median 86% decrease; mean 85%) and up to 89% depletion of monocytic (CD14+Lin-HLA-DR-/low; median 33% decrease; mean 45%) MDSCs. Treatment resulted in a significant increase in activated CTLs (PD-1+GITR+ CD8+ T-cells as a proportion of all CD8+ T-cells) in 5/6 evaluable patients (median 322% increase; mean 352%). Peak effects on ApoE, MDSCs, and CTLs began ~2 weeks after RGX-104 initiation. In order to maximize the achievement of biologically relevant RGX-104 exposure, and therefore desirable immune effects throughout the treatment period, continuous dosing at 120 mg BID is being evaluated in cohort 3. One of two evaluable patients in cohort 3 experienced reversible grade 4 neutropenia and grade 2 lipid elevations, which are “on-target” adverse events. No other grade 3-4 adverse events attributable to RGX-104 have occurred. One renal cancer and one melanoma patient-both refractory to anti-PD-1 therapy–had stable disease. Conclusions: Proof of principle has been demonstrated for the immune-stimulatory action of the first-in-class LXR-agonist RGX-104 in advanced cancer patients. RGX-104 activated ApoE in peripheral blood cells in a dose-dependent fashion and was well tolerated when administered orally at 120 mg or 240 mg QD. RGX-104 selectively depleted MDSCs, leading to activation of the relevant PD-1+ CTL population in a diverse group of cancer patients harboring PD-1 inhibitor refractory disease and immune-therapy resistant cancers. Dosing of RGX-104 at 120 mg BID continues. Future plans include studying RGX-104 both as monotherapy and in combination with a PD-1 inhibitor in select malignancies. Citation Format: Monica Mita, Alain Mita, Bartosz Chmielowski, Michael Postow, Erika Hamilton, Shubham Pant, Roger Waltzman, Eric Rowinsky, Michael Szarek, Foster Gonsalves, Isabel Kurth, Celia Andreu, Ilana Pollack, Daniel Mucida, Raissa Tanqueco, Sohail Tavazoie, Masoud Tavazoie. A phase 1 trial of RGX-104, a first-in-class immunotherapy targeting the liver-X nuclear hormone receptor (LXR), in patients with refractory malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B001.

A Combined In Vivo HSC Transduction/Selection Approach Results in Efficient and Stable Gene Expression in Peripheral Blood Cells in Mice

We recently reported on an in vivo hematopoietic stem cell (HSC) gene therapy approach. It involves the subcutaneous injections of G-CSF/AMD3100 to mobilize HSCs from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating helper-dependent adenovirus vector system. HSCs transduced in the periphery homed back to the bone marrow, where they persisted long-term. However, high transgene marking rates found in primitive bone marrow HSCs were not reflected in peripheral blood cells. Here, we tested small-molecule drugs to achieve selective mobilization and transduction of HSCs. We found more efficient GFP marking in bone marrow HSCs but no increased marking in the peripheral blood cells. We then used an in vivo HSC chemo-selection based on a mutant of the O6-methylguanine-DNA methyltransferase (mgmtP140K) gene that confers resistance to O6-BG/BCNU and should give stably transduced HSCs a proliferation stimulus and allow for the selective survival and expansion of progeny cells. Short-term exposure of G-CSF/AMD3100-mobilized, in vivo-transduced mice to relatively low selection drug doses resulted in stable GFP expression in up to 80% of peripheral blood cells. Overall, the further improvement of our in vivo HSC transduction approach creates the basis for a simpler HSC gene therapy.

Expression level of CXCL7 in peripheral blood cells is a potential biomarker for the diagnosis of renal cell carcinoma.

There are no blood biomarkers for the diagnosis of renal cell carcinoma (RCC) in routine clinical use. We focused on the gene expression profile of peripheral blood cells obtained from RCC patients to discover novel biomarkers for RCC diagnosis. Using microarray analysis and quantitative verification, CXCL7 was shown to be significantly upregulated in the peripheral blood cells of RCC patients. Importantly, aberrant CXCL7 expression was confirmed even in peripheral blood cells obtained from early stage (pT1a) RCC patients, and the expression level of CXCL7 in peripheral blood cells was a potential independent biomarker for the diagnosis of RCC by receiver operating characteristic curve analysis (sensitivity, 70.0%; specificity, 64.0%; area under the curve = 0.722; multiple logistic regression analysis: odds ratio, 1.07; 95% confidence interval, 1.03–1.11; P = 0.0004). Moreover, CXCL7 expression in peripheral blood cells significantly decreased after resection of the primary tumor. CXCL7 is more highly expressed in PBMCs than in neutrophils from both healthy controls and RCC patients. Interestingly, CXCL7 expression in PBMCs from healthy volunteers was significantly elevated following coculture with RCC cells compared to those cocultured with normal cells as a control. These results suggest that aberrant CXCL7 expression in peripheral blood cells is induced by RCC cells and may serve as a novel biomarker in the diagnosis of RCC.