2-ΔΔCt Value Research Articles

Biomarker-Guided Drug Repurposing and Molecular Validation of Angiotensin-2 Receptor Type-1 in Brain Tumor

Glioma, the most frequent and malignant brain tumor, is one of the most lethal forms of cancer. Among a wide range of aberrantly expressed genes, Angiotensin-2 Receptor Type-1 (AGTR1) is reported to be up-regulated in glioma and associated with aggressive tumor traits and progression. The current study evaluates the anticancer potential of commonly used angiotensin receptor blocker (ARB) telmisartan. A total of eleven, Food and Drug Administration (FDA) approved ARBs were selected, and eight drugs were docked against the AGTR1 receptor. The antihypertensive drug with the highest docking score was selected for in vitro experimentation. Half maximal inhibitory concentration was determined and subsequently applied to patient-derived glioma cell lines. Quantitative gene expression post-drug treatment was determined through real-time polymerase chain reaction (PCR). In vitro growth inhibitory assays revealed that telmisartan at a dose of 45±0.06 μM was able to inhibit 50% of the cell population in malignant glioma U87 cell lines. The PCR results show that AGTR1 expression in the untreated sample was high, as evidenced by 2-ΔΔCt values (342.4, 138.5, 1467.3) for sample IK148-Glioblastma multiform (GBM), IK163-low grade glioma (LGG), and IK231-Medulloblastoma (MDB), respectively. The comparison was made with positive control U-87 cell lines. Following the drug treatment, an increase in the cycle threshold (Ct) mean value for AGTR1 was observed, coinciding with a decrease in mean fold changes of 0.06±0.8, 0.5±2.0, and 0.02±0.02 for GBM, LGG, and MDB samples, respectively. Based on the findings of this study, it can be concluded that telmisartan exhibits successful inhibitory effects against AGTR1 expression in glioma cell lines.

POTENTIAL BIOMARKERS FOR EARLY DETECTION OF ORAL CANCER: LEPTIN AND ADIPONECTIN

Aim: To determine adiponectin and leptin expression levels in samples from healthy, dysplastic and cancerous oral lesions and to investigate the potential role of these biomarkers in oral cancer development. 
 Methods: Tissue samples from the archives which were histologically diagnosed as oral squamous cell carcinoma (OSCC) (n=24), oral dysplasia (n=12) and healthy tissue (n=16) were included. The concentration and purity of samples were determined after total RNA extraction. Thereafter, cDNA synthesis was performed. The differences between expression levels were evaluated with qPCR. Expression levels related to OSCC were performed according to normalizing gene ACTB. 2-ΔΔCT method was used for comparison of expression levels. Student t-test was used to compare the differences in gene expression levels between groups, using 2-ΔΔCT value for each gene. 
 Results: Adiponectin and leptin expression levels were decreased by 1.09 (p = 0.49) and 10.3 (p = 0.22), respectively, in OSCC group, however differences were not statistically significant. The level of adiponectin expression decreased by 1.65 times (p = 0.62) and leptin expression increased by 3 times (p = 0.30) in the dysplasia group. When OSCC and dysplasia groups were compared, it was found that the level of adiponectin expression increased by 1.52 times (p = 0.99) and the level of leptin expression decreased by 30.94 times (p = 0.16) in the OSCC group. The samples were re-grouped as increased risk (OSCC+dysplasia) (n=36) and healthy (n=16) and a decrease by 16.5 (p= 0.3) in leptin expression and an increase by 1.14 (p= 0.6) in adiponectin expression was observed in the increased risk group compared to the healthy group.
 Conclusion: In tissue samples diagnosed as healthy, dysplastic and OSCC, expression levels for leptin and adiponectin have not been compared before. The results revealed a significant decrease in leptin expression and an increase in adiponectin expression in increased risk group.

P53 Pathway Activation Mediated High c-MAF Expression Is Associated with Overall and Post-Progression Survival in Multiple Myeloma

Background: Chromosomal abnormalities are strongly associated with prognosis of multiple myeloma (MM). Among them, t (14;16) resulting in high expression of c-MAF, and t (14;20) resulting in high expression of MAFB, lead to poor prognosis. However, clinical significance and mechanisms underlying high c-MAF and MAFB expression without these translocations have not been fully elucidated. Methods: A total of 96 MM and 38 MGUS patients, 10 controls, and 9 MM cell lines were included in this study. RNA was extracted from purified CD138+ plasma cells from bone marrow (BM) mononuclear cells. c-MAF, MAFB, p53, and p21 RNA expressions were determined by RQ-PCR. Their expression levels were normalized against ACTB levels and calculated with 2-ΔΔCt value. Inhibition studies using BRD4 inhibitor JQ1, and MYC inhibitor10058-F4, and MM cell lines expressing doxycycline-inducible p53 (Tet-on p53) or c-MAF knockdown (KD) with siRNA were used for in vitro studies. Results: c-MAF expression in MM patients was higher than in MGUS patients and control (median level 0.043, 0.025, 0.002, p<0.001), but MAFB expression did not differ between MM, MGUS, and control (p=0.371). Although c-MAF expression level was significantly higher in MM with t (14;16) (p=0.0018), high c-MAF expression was observed in patients without the translocations, suggesting a role of c-MAF in MM progression. Other cytogenetic abnormalities such as del 17p, or t (4;14), did not affect c-MAF expression. Overall survival (OS) of MM patients with high c-MAF expression was significantly inferior compared to the patients with low c-MAF expression (HR 2.46, p=0.002, 2 years survival rate 42.9% vs. 72.7%, median 20.3 months vs. not reached), but progression-free survival (PFS) did not differ (p=0.551). Instead, post-progression survival (PPS) was significantly shorter in the patients with high c-MAF expression (HR 4.67, p<0.001, two years survival 0% vs. 60.2%, median 3.6 months vs. 49.3 months) suggesting that c-MAF confers drug resistance to residual disease. MAFB expression did not affect OS, PFS, and PPS. Reduction of cyclin D2 expression by c-MAF KD in vitro and a positive correlation between c-MAF and cyclin D2 expression (r=0.29, p<0.001) in vivo in patients, support previous reports describing that MM proliferation is induced by c-MAF via cyclin D2. c-MAF was not correlated with APOBEC3B expression. c-MAF expression was positively correlated with MDM2 and MYC (r=0.387, p=0.03 and r=0.221, p=0.019, respectively) but not with p53 and PUMA. A tendency of positive correlation was also observed with p21/CDK1NA (r=0.304, p=0.085). p53 overexpression from the Tet-on p53 and p53 stabilization by nutlin-3 increased c-MAF expression in three cell lines with t (14;16). c-MAF KD together with p53 overexpression significantly suppressed the proliferation, implying synthetic lethality. These findings suggest that c-MAF expression is upregulated in response to proliferation arrest induced by the p53 pathway; suppressing this responsive c-MAF expression abrogates cell growth more efficiently. JQ1 and CPI203 increased c-MAF expression in cell lines with t (14;16), suggesting that c-MAF expression is not controlled by super-enhancers. Conclusion: Although the mechanism of high expression of c-MAF and MAFB has previously been reported in IgH translocation, the relationship between c-MAF and drug resistance remains to be determined. The induction of c-MAF expression by tumor suppressor gene p53 is suggested to be a mechanism underlying poor prognosis of MM with t (14;16), and suppressing c-MAF expression with chemotherapeutic drugs might prevent emergence of drug resistance in residual tumor cells. Disclosures Handa: Ono: Research Funding.

DNA Polymerases Pol θ/Pol η Involved in Error-Prone DNA Repair Are Highly Expressed in Multiple Myeloma and Upregulated By DNA Damage

Background: DNA polymerases (DNA pols) are essential enzymes for DNA replication. In mammalian cells, DNA pols are divided into four families: A (Pol θ, Pol γ, and Pol ν), B (Pol α, Pol δ, Pol ε, and Pol ζ), X (Pol β, Pol λ, Pol μ, and TDT), and Y (Pol η, Pol ι, Pol κ, and REV1). These DNA pols are required for both genome duplication and protecting cells from DNA damage induced by endogenous and exogenous agents, such as ROS, UV, and chemotherapeutic drugs. For example, Pol β, Pol λ, and Pol ι participate in base excision repair. Contrastingly, Pol ζ, REV1, Pol η, Pol ι, and Pol κ can replicate over various DNA lesions to prevent DNA replication stalling, known as translesion synthesis. Although some DNA pols are highly expressed in cancer cells, indicating chemotherapeutic resistance and poor outcome, their exact roles and expression mechanisms have not been fully elucidated. Multiple myeloma (MM) is a hematological malignancy of terminally differentiated plasma cells, with multistep progression from pre-cancer stage namely. In this study we attempted to elucidate the involvement of DNA pols in multistep oncogenesis of MM. Methods: A total of 63 MM and 29 MGUS patients, 15 controls, and 9 MM cell lines were included in the study. RNA was extracted from purified CD138+ plasma cells. DNA pol expressions were determined by RQ-PCR. Their expression levels were normalized against ACTB levels and calculated with 2-ΔΔCt value. Doxycycline-inducible p53 system (Tet-on p53) and nutlin-3 were used for analyzing the role of p53 in DNA pol expressions in MM cell lines. Melphalan, doxorubicin, and bortezomib were used to examine DNA pol expressions in damaged cells in vitro. JQ1 and CPI203 were used to evaluate the role of bromodomain in DNA pol expressions. Results: Pol α and Pol ε expressions were significantly higher in MM than in control (p=0.007 and p=0.004, respectively), but Pol ε and Pol ζ levels were not significantly different (p=0.631, p=0.0826, respectively). Pol η, REV1, Pol ι, and Pol κ expressions were significantly higher in MM than control (p<0.001, p=0.002, p<0.001, and p<0.001, respectively). Pol θ and Pol γ were expressed at a higher level in MM than in control (p<0.001 and p<0.001, respectively). Pol β and Pol λ expressions were higher in MM than in control (p=0.0088 and p=0.013, respectively). Although the expressions of many DNA pols were higher in MM plasma cells, we focused on Pol η and Pol θ, because Pol λ, Pol μ, Pol ν, and Pol ι were expressed at very low levels, and Pol ε, Pol ζ, Pol γ, Pol κ, and REV1 were expressed in PBMNCs of healthy volunteers at high level. Pol η and Pol θ expressions did not differ due to known risk factors, such as cytogenetic abnormalities and ISS. Pol η expressions were positively correlated with p53 and myc expressions (r=0.718, p<0.001, r=0.528, p<0.001 respectively). p53 overexpression by Tet-on vector or nutlin-3 treatment enhanced Pol η expression, indicating that Pol η expression is regulated by p53. Melphalan or doxorubicin increased Pol η expression, but bortezomib or lenalidomide did not, suggesting that Pol η is upregulated by DNA damage via p53 pathway. Overall survival of the patients with high Pol η expression tended to be worse than with low Pol η expression (24 months survival: 69.6% vs. 57.9%, p=0.29). Pol θ expression was weakly correlated with p53. Melphalan induced Pol θ expression but doxorubicin did not. JQ1 significantly reduced Pol θ expression suggesting that Pol θ was regulated by bromodomain. Conclusion: We found that Pol θ and Pol η are highly expressed in MM, and upregulated by DNA damage. These DNA pols are involved in drug resistance and genomic instability leading to poor prognosis. Thus, DNA pols can be used as novel therapeutic targets and prognostic markers. Disclosures Handa: Ono: Research Funding.

Abstract 3125: Functional validation of respective gene from a nineteen gene-based risk score classifier, TCA19 as a prognostic indicator and response to immune system of colorectal cancer patients

Abstract Background: Colorectal cancer (CRC) is a leading cause of cancer death worldwide. Pathologic staging is the gold standard for prognosis, concurrently selecting patients for adjuvant chemotherapy. A recent study reports that the risk score based 19 gene regulated by TREM1 or CTGF activation, TCA19, was an independent risk factor for disease-free survival. Objective: The aim of this study was to validate TCA19 as a prognostic indicator of colorectal cancer patients and to select candidate genes related to clinicopathologic variables and immune system. Patients and Method: A retrospective review was conducted on patients who underwent surgery followed by adjuvant chemotherapy for CRC at the Asan Medical Center, Seoul, Korea, between December 2008 and December 2014. To validate TCA19, the medical records of 60 patients who met inclusion criteria were reviewed. According to the previous report, RNA sequencing using reverse transcription-quantitative polymerase chain reaction (RT-PCR) was done and then TCA19 risk score using 2ΔΔCt values was checked. After checking TCA19 genes, we selected key candidate genes according to the correlation with clinicopathologic variables and PFS. Immunohistochemistry (IHC), Western blot, and immunofluorescence staining (IFS) were done for the selected candidate genes, TREM1, CKS2, SLAMF7. Results: Among 60 study cohort, 59 patients were in stage IV and 1 patient was in stage IIIC. Among 30 patients with high TCA risk score, the PFS rates between 4 patients who received 5-FU regimen and 8 patients received the target regimens (bevacizumab or cetuximab) were significantly different. On multivariate analyses for PFS, lymphovascular invasion (LVI), circumferential resection margin (CRM), SLAMF7, CKS2, and TREM1 were risk factors associated with PFS. Based on the association with clinicopathologic variables and the survival results, the SLAMF7, the CKS2, and the TREM1 can become a key gene among the TCA 19 genes in terms of prognostic factor and previous results. Primary and metastatic tumor IHC of the SLAMF7, the CKS2, and the TREM1 was significantly overexpressed, compared with normal tissue IHC of the three genes. Like IHC results, 5 days' IFS imaging of SLAMF7 after co-culture showed significant hypoexpression compared with 0 day imaging of SLAMF7. Conclusion: TCA 19 risk score might be one of prognostic predictors related to using the target regimens (bevacizumab or cetuximab). TREM1, CKS2, and SLAMF7 were key candidate genes among the TCA19 genes in terms of clinicopathologic variables and survival outcomes. TREM1 was overexpressed in primary or metastatic tumor on the contrary; downregulation of SLAMF7 was shown in the primary or metastatic tumor. Further functional study using a large cohort will be needed. Citation Format: Jong Lyul Lee, Jin Cheon Kim, Chan Wook Kim, Seon Ae Roh. Functional validation of respective gene from a nineteen gene-based risk score classifier, TCA19 as a prognostic indicator and response to immune system of colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3125.

Immunogenicity of a live attenuated classical swine fever vaccine in pigs under field conditions

A study was conducted for assessing the immunogenicity of live classical swine fever (CSF) vaccine in pigs under field conditions. Pre immunization blood samples and post immunization (PI) blood samples at 30, 60, 90, and 120 days post immunization (DPI) were collected. Th1 response was assessed by the expression of IFN-γ, IL- 2 and IL-12, and serum antibody titres and IL-10 expression were studied for Th2 response. The mean±SE of IFN- γ gene response as fold change (2ΔΔCt values) at 30, 60, 90, and 120 days post immunization (DPI) were 9.61±0.65, 3.49±0.44, 2.22±0.407, and 3.12±1.35 respectively. IFN-γ response showed significantly increased fold change during PI period. Significant peak increase in the expression level was observed at 30 DPI. IL-2 gene response showed significantly increased fold change during PI (Mean expression at 30, 60, 90, and 120 DPI was 4.41, 2.61, 3.291 and 2.09 respectively) and peak increase was observed at 30 DPI. IL-12 response also showed significant increase during PI with a peak expression at 30 DPI (Mean fold changes at 30, 60, 90, and 120 days post immunization were 4.92, 2.97, 2.72, and 3.61 respectively). Maximum IL-10 expression was observed at 60 DPI (Mean fold change at 30, 60, 90, and 120 days post immunization were 3.364±0.723, 11.56±1.64, 4.73±1.01, and 2.599±1.05 respectively). Pre- and post-immunization antibody responses were monitored by enzyme linked immunosorbent assay. Antibody titres increased consistently from 30 days after primary immunization until 120 days. Montanide (gel 01) coupled PK-15 adapted live, CSV vaccine induced a good Th1 response followed by Th2 response.

β2-microglobulin gene duplication in cetartiodactyla remains intact only in pigs and possibly confers selective advantage to the species.

Several β2-microglobulin (B2M) -bound protein complexes undertake key roles in various immune system pathways, including the neonatal Fc receptor (FcRn), cluster of differentiation 1 (CD1) protein, non-classical major histocompatibility complex (MHC), and well-known MHC class I molecules. Therefore, the duplication of B2M may lead to an increase in the biological competence of organisms to the environment. Based on the pig genome assembly SSC10.2, a segmental duplication of ~45.5 kb, encoding the entire B2M protein, was identified in pig chromosome 1. Through experimental validation, we confirmed the functional duplication of the B2M gene with a completely identical coding sequence between two copies in pigs. Considering the importance of B2M in the immune system, we performed the phylogenetic analysis of B2M duplication in ten mammalian species, confirming the presence of B2M duplication in cetartioldactyls, like cattle, sheep, goats, pigs and whales, but non-cetartiodactyl species, like mice, cats, dogs, horses, and humans. The density of long interspersed nuclear element (LINE) at the edges of duplicated blocks (39 to 66%) was found to be 2 to 3-fold higher than the average (20.12%) of the pig genome, suggesting its role in the duplication event. The B2M mRNA expression level in pigs was 12.71 and 7.57 times (2-ΔΔCt values) higher than humans and mice, respectively. However, we were unable to experimentally demonstrate the difference in the level of B2M protein because species specific anti-B2M antibodies are not available. We reported, for the first time, the functional duplication of the B2M gene in animals. The identification of partially remaining duplicated B2M sequences in the genomes of only cetartiodactyls indicates that the event was lineage specific. B2M duplication could be beneficial to the immune system of pigs by increasing the availability of MHC class I light chain protein, B2M, to complex with the proteins encoded by the relatively large number of MHC class I heavy chain genes in pigs. Further studies are necessary to address the biological meaning of increased expression of B2M.

Circulating tumour cells as biomarkers for evaluating cryosurgery on unresectable hepatocellular carcinoma.

We evaluated the efficacy of pre-cryosurgery and post-cryosurgery circulating tumour cells (CTCs) as biomarkers for unresectable hepatocellular carcinoma (HCC). Real‑time qPCR was used to detect potential biomarker genes in CTCs, and magnetic-activated cell sorting (MACS) and fluorescence‑activated cell sorting (FACS) was performed on 47patients with hepatocellular cancer who underwent cryosurgery. CTCs in the 47patients were assessed 1day before cryosurgery, and 7and30days after cryosurgery. The number of CTCs was 17.70±5.725, 14.64±6.761 and 10.28±5.598, respectively, and this decreased significantly over time(P<0.01). ΔCt values for MAGE-3, survivin and carcinoembryonic antigen (CEA) were elevated significantly compared with those obtained before cryosurgery; 2-ΔΔCt values were <1 before cryosurgery, and were 0.63±1.56, 0.21±0.22 and 0.22±0.34 for MAGE-3, survivin and CEA, respectively, at 7days after treatment. At 30days after treatment, 2-ΔΔCt values for MAGE-3, survivin and CEA were 0.24±0.82, 0.03±0.07 and 0.02±0.08, indicating that gene expression in CTCs significantly decreased over time (P<0.01). CTCs were useful biomarkers for evaluating the efficacy of cryosurgery on unresectable HCC.

Mesenchymal STEM CELLS Favor Tumor Growth By Generating Granulocyte-like Myeloid Derived Suppressor CELLS in CML Patients

INTRODUCTION. The complex interplay between cancer cells and immune system allows neoplastic cells to evade immune surveillance and expand. Recently, our and another group have demonstrated that a subpopulation of myeloid cells, defined as "granulocytic myeloid-derived suppressor cells" (G-MDSC), plays an important role for immune escape in chronic myeloid leukemia (CML) patients by reducing T cell activation. The aim of this study was to evaluate the influence of Mesenchymal stem cells (MSC) on generation of MDSCs by comparing CML MSCs (n=10) with healthy donors (HD) MSC (n=8).METHODS. G-MDSC (CD11b+CD33+CD14-HLADR- cells) were analyzed in peripheral blood (PB) of 20 healthy donors (HD) and 30 CML patients at diagnosis by cytofluorimetric analysis. Immuno-suppressive activity was tested through incubation of G-MDSC with autologous CFSE-labeled T cells and stimulation with phytohaemagglutinin (PHA). Controls included a positive T cell proliferation control (T cells plus PHA) and a negative one (T cells only). After three days, T cell proliferation was analyzed by flow cytometry. For G-MDSC generation, human peripheral blood mononucleated cells (PBMC) from HD were cultured alone and with MSC of CML (n=10) or HD (n=8) (1:100 ratio). After one week, G-MDSC were isolated using anti-CD66b magnetic microbeads and the phenotype was confirmed by cytofluorimetric analysis. Expression of ARG1, NOS2, PTGS2, TNFα, TGFβ, IL6, IL10, IL1β was also evaluated using real time PCR.RESULTS. Percentage of cells with a G-MDSC phenotype was greater in PB obtained from CML patients than HD (82.5±9.6% vs 56,2±5.4%, p<0.0001). G-MDSC were able to inhibit T cell proliferation compared to positive control (25±5% vs 48±7.6%, p=0.0057). To investigate if CML MSC may be involved in G-MDSC generation, we incubated HD PBMC with CML or HD MSC for one week. After magnetic isolation, we found that only CML MSC-educated G-MDSC acquired immune-suppressive ability, inhibiting T cell proliferation compared to G-MDSCs control (isolated from PBMC cultured in medium alone) (32±12% vs 63±5.9%, p=0.003). On the contrary, HD MSC-educated G-MDSC did not show any suppressive effect. We also found that CML MSC-educated G-MDSC expressed higher level of the following immune modulatory factors: TNFα (20.8±19.3, p=0.006), IL1β (47.3±25.2, p=0.001), PTGS2 (20.7±10.9, p=0.002) and IL6 (33.8±13.9, p=0.004) compared to HD MSC-educated G-MDSCs (arbitrarily 2-ΔΔCt value: 1). MSC WE also observed ane an up-regulation of PTGS2 (19±4.4, p=0.04), TGFβ (6±3, p=0.01) and IL6 (5±2.8, p=0.04) in CML MSCs at time 0 with a great variability among the patients (calculated value of 2-ΔΔCt in HD MSC was 1). After 48 h of co-culture with PBMC, CML MSC showed statistically significant up-regulation of ARG1 (23.5±11.9, p=0.02), TGFβ (4.8±3, p=0.04), IL10 (5.6±2.8, p=0.03) and IL6 (54.3±23, p=0.02) expression, suggesting that multiple mechanisms are involved in MDSC induction by CML MSC.CONCLUSION. Our work demonstrates that CML MSCs are able to activate MDSCs favoring cancer immune evasion in CML patients. DisclosuresPalumbo:Novartis: Honoraria, Other: Advisory Board.

Quantitative measurement of serum microRNA-21 expression in relation to breast cancer metastasis in Chinese females.

BackgroundBreast cancer is the most common type of cancer in females. Aberrant expression of microRNA-21 (miR-21) has previously been reported in breast cancer tissue. The aim of this study was to investigate expression levels of serum miR-21 in breast cancer patients and evaluate its prognostic value in Chinese females.MethodsReal-time quantitative (RQ)-PCR was used to analyze miR-21 expression in archived serum, tumor tissue, and adjacent normal tissue from 549 participants (326 with breast cancer, 223 without breast cancer). We also analyzed associations between serum miR-21 expression and breast cancer subtypes and patient prognosis. Recurrence and survival were analyzed by using the multivariate Cox proportional hazards model.ResultsExpression of miR-21 was significantly higher in breast cancer tissues compared with normal adjacent breast tissues (P<0.001). The 2-ΔΔCt values for serum miR-21 in breast cancer patients versus healthy controls were 9.12±3.43 and 2.96±0.73, respectively. Multivariate Cox proportional hazards model suggested that serum miR-21 expression was an independent poor prognostic factor for both recurrence (hazard ratio [HR]= 2.942; 95% confidence interval [CI]=1.420-8.325; P=0.008) and disease-free survival (HR=2.732; 95% CI=1.038-7.273, P=0.003) in breast cancer.ConclusionsIncreased serum miR-21 expression level was correlated with poor prognosis of breast cancer patients, indicating that serum miR-21 may be a novel prognostic marker for recurrence and survival of breast cancer patients before resection.

Effects of Human Insulin Gene Transfection on the Adipogenic Differentiation of Human Umbilical Cord Mesenchymal Stem Cells in Silk Fibroin Scaffolds &amp;lt;i&amp;gt;in Vitro&amp;lt;/i&amp;gt;

The resorption of the transplanted fat over time limited the use of autologous fat for the reconstruction of soft tissue defect. Tissue engineering (TE) adipose with silk fibroin scaffold could be a promising substitute for soft tissue filling. In this study, we try to develop a tissue engineering adipose in vitro by seeding silk fibroin scaffold with human umbilical cord mesenchymal stem cells (hUCMSCs) after transfected with recombinant human insulin gene lentivirus. Our aim was to observe the effects of the insulin gene transfection on the adipogenesis of hUCMSCs when cultured with silk fibroin scaffolds. The hUCMSCs infected with recombinant lentiviral pLenti6.3-insulin-IRES-EGFP were seeded on silk fibroin scaffolds and cultured in adipogenic differentiation medium for 5 - 7 days. The expression of adipogenic gene PPARγ-2 was tested by RT-PCR after 7 days culture of adipogenic induction. The accumulation of cytoplasmic droplets of neutral lipids was assessed by Oil Red O staining. The RNA and protein expression of transfected insulin gene in hUCMSCs were detected by QPCR and western blot. The effect of recombinant lentivirus transfection on the growth and proliferation of hUCMSCs was observed by MTT test. We observed that the 2-ΔΔCt value of insulin gene expression of hUCMSCs in the transfected group was 300.25 times higher than that in the untransfected group. The western blot showed that a positive band was discerned at the site of a relative molecular mass of 8 × 103 Dalton in transfected group. After adipogenic culture for 7 days, under the fluorescent inverted phase-contrast microscope, after Oil Red O staining, a lot of adipocytes appeared in silk fibroin scaffold; round adipose droplets showed intracellularly; the size of the adipocytes was not homogenous, and the density of adipocytes in transfected group was significantly higher than that in untransfected group (P = 0.007, P 0.05). And there was also no significant difference in optical density (A) between cell group and cell-scalffold group (P = 0.066, P > 0.05). We concluded that insulin gene could obviously promote the adipogenic differentiation of hUCMSCs, and a tissue engineering adipose could be constructed by the silk fibroin scaffolds seeded with human insulin gene-modified hUCMSCs effectively in vitro.

Gene‐diet interaction effects during a supplementation with n‐3 PUFA on both plasma and gene expression levels of inflammatory markers (1037.9)

Objective: To test whether gene expression of inflammatory genes is altered following an n‐3 PUFA supplementation and to test for any possible gene‐diet interactions modulating plasma inflammatory biomarker levels. Methods: 210 subjects completed a 6‐wk n‐3 PUFA supplementation with 5g/d of fish oil. Gene expression of TNF and IL6 was assessed in peripheral blood mononuclear cells (PBMC) using the TaqMan technology adjusted for the endogenous control (GAPDH). Genotyping of 20 SNPs from the TNF‐LTA gene cluster, IL1β, IL6 and CRP genes was performed. Results: Gene expression of TNF and IL6 increased after the 6‐wk n‐3 PUFA supplementation as shown by 2‐ΔΔCt values &gt;1 (mean±SD, 1.05±0.39 and 1.18±0.50, respectively). In a MIXED model for repeated measures adjusted for the effects of age, sex and BMI, gene by supplementation interactions effects were observed for rs1143627, rs16944, rs1800797, and rs2069840 on IL6 levels, for rs2229094 on TNF‐alpha levels and for rs1800629 on CRP levels (p&lt;0.05 for all). Conclusions: An n‐3 PUFA supplementation could alter gene expression levels of TNF and IL6 in PBMC. Also, the large inter‐individual variability in plasma inflammatory markers following an n‐3 PUFA supplementation may be influenced by genetic variations in inflammatory genes.Grant Funding Source: CIHR

Altered Immune Response in Mice Deficient for the G Protein-coupled Receptor GPR34

The X-chromosomal GPR34 gene encodes an orphan G(i) protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-α, GM-CSF, and IFN-γ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges.