The time-dependent expression of FPR2 and ANXA1 in murine deep vein thrombosis model and its relation to thrombus age.

Thrombus age determination in fatal venous thromboembolism cases is an important task for forensic pathologists. In this study, we investigated the time-dependent expressions of formyl peptide receptor 2 (FPR2) and Annexin A1 (ANXA1) in a stasis-induced deep vein thrombosis (DVT) murine model, with the aim of obtaining useful information for thrombus age timing. A total of 75 ICR mice were randomly classified into thrombosis group and control group. In thrombosis group, a DVT model was established by ligating the inferior vena cava (IVC) of mice, and thrombosed IVCs were harvested at 1, 3, 5, 7, 10, 14, and 21 days after modeling. In control group, IVCs without thrombosis were taken as control samples. The expressions of FPR2 and ANXA1 during thrombosis were detected using immunohistochemistry and double immunofluorescence staining. Their protein and mRNA levels in the samples were determined by Western blotting and quantitative real-time PCR. The results reveal that FPR2 was predominantly expressed by intrathrombotic neutrophils and macrophages. ANXA1 expression in the thrombi was mainly distributed in neutrophils, endothelial cells of neovessels, and fibroblastic cells. After thrombosis, the expressions of FPR2 and ANXA1 were time-dependently up-regulated. The percentage of FPR2-positive cells and the level of FPR2 protein significantly elevated at 1, 3, 5 and 7 days after IVC ligation as compared to those at 10, 14 and 21 days after ligation (p < 0.05). Moreover, the mRNA level of FPR2 were significantly higher at 5 days than that at the other post-ligation intervals (p < 0.05). Besides, the levels of ANXA1 mRNA and protein peaked at 10 and 14 days after ligation, respectively. A significant increase in the mRNA level of ANXA1 was found at 10 and 14 days as compared with that at the other post-ligation intervals (p < 0.01). Our findings suggest that FPR2 and ANXA1 are promising as useful markers for age estimation of venous thrombi.

Time-Dependent Sequential Changes of IL-10 and TGF-ß1 in Mice with Deep Vein Thrombosis.

OBJECTIVES: To detect the expression changes of interleukin-10 (IL-10) and transforming growth factor-ß1 (TGF-ß1) during the development of deep vein thrombosis in mice, and to explore the application value of them in thrombus age estimation. METHODS: The mice in the experimental group were subjected to ligation of inferior vena cava. The mice were sacrificed by excessive anesthesia at 1 d, 3 d, 5 d, 7 d, 10 d, 14 d and 21 d after ligation, respectively. The inferior vena cava segment with thrombosis was extracted below the ligation point. The mice in the control group were not ligated, and the inferior vena cava segment at the same position as the experimental group was extracted. The expression changes of IL-10 and TGF-ß1 were detected by immunohistochemistry (IHC), Western blotting and real-time qPCR. RESULTS: IHC results revealed that IL-10 was mainly expressed in monocytes in thrombosis and TGF-ß1 was mainly expressed in monocytes and fibroblast-like cells in thrombosis. Western blotting and real-time qPCR showed that the relative expression levels of IL-10 and TGF-ß1 in each experimental group were higher than those in the control group. The mRNA and protein levels of IL-10 reached the peak at 7 d and 10 d after ligation, respectively. The mRNA expression level at 7 d after ligation was 4.72±0.15 times that of the control group, and the protein expression level at 10 d after ligation was 7.15±0.28 times that of the control group. The mRNA and protein levels of TGF-ß1 reached the peak at 10 d and 14 d after ligation, respectively. The mRNA expression level at 10 d after ligation was 2.58±0.14 times that of the control group, and the protein expression level at 14 d after ligation was 4.34±0.19 times that of the control group. CONCLUSIONS: The expressions of IL-10 and TGF-ß1 during the evolution of deep vein thrombosis present time-dependent sequential changes, and the expression levels of IL-10 and TGF-ß1 can provide a reference basis for thrombus age estimation.

ADRM1-amplified metastasis gene in gastric cancer.

The proteasome ubiquitin receptor ADRM1 has been shown to be a driver for 20q13.3 amplification in epithelial cancers including ovarian and colon cancer. We performed array-CGH on 16 gastric cancer cell lines and found 20q13.3 to be amplified in 19% with the minimal amplified region in gastric cancer cell line AGS spanning a 1 Mb region including ADRM1. Expression microarray analysis shows overexpression of only two genes in the minimal region, ADRM1 and OSBPL2. While RNAi knockdown of both ADRM1 and OSBPL2 led to a slight reduction in growth, only ADRM1 RNAi knockdown led to a significant reduction in migration and growth in soft-agar. Treatment of AGS cells with the ADRM1 inhibitor RA190 resulted in proteasome inhibition, but RNAi knockdown of ADRM1 did not. However, RNAi knockdown of ADRM1 led to a significant reduction in specific proteins including MNAT1, HRS, and EGFR. We hypothesize that ADRM1 may act in ADRM1-amplified gastric cancer to alter protein levels of specific oncogenes resulting in an increase in metastatic potential. Selective inhibition of ADRM1 independent of proteasome inhibition may result in a targeted therapy for ADRM1-amplified gastric cancer. In vivo models are now warranted to validate these findings. © 2015 Wiley Periodicals, Inc.

The HPV16E7 Affibody as a Novel Potential Therapeutic Agent for Treating Cervical Cancer Is Likely Internalized through Dynamin and Caveolin-1 Dependent Endocytosis.

Affibodies targeting intracellular proteins have a great potential to function as ideal therapeutic agents. However, little is known about how the affibodies enter target cells to interact with intracellular target proteins. We have previously developed the HPV16E7 affibody (ZHPV16E7384) for HPV16 positive cervical cancer treatment. Here, we explored the underlying mechanisms of ZHPV16E7384 and found that ZHPV16E7384 significantly inhibited the proliferation of target cells and induced a G1/S phase cell cycle arrest. Furthermore, ZHPV16E7384 treatment resulted in the upregulation of retinoblastoma protein (Rb) and downregulation of phosphorylated Rb (pRb), E2F1, cyclin D1, and CDK4 in the target cells. Moreover, treatment with dynamin or the caveolin-1 inhibitor not only significantly suppressed the internalization of ZHPV16E7384 into target cells but also reversed the regulation of cell cycle factors by ZHPV16E7384. Overall, these results indicate that ZHPV16E7384 was likely internalized specifically into target cells through dynamin- and caveolin-1 mediated endocytosis. ZHPV16E7384 induced the cell cycle arrest in the G1/S phase at least partially by interrupting HPV16E7 binding to and degrading Rb, subsequently leading to the downregulation of E2F1, cyclin D1, CDK4, and pRb, which ultimately inhibited target cell proliferation. These findings provide a rationale of using ZHPV16E7384 to conduct a clinical trial for target therapy in cervical cancer.

Effect of different intravenous iron preparations on lymphocyte intracellular reactive oxygen species generation and subpopulation survival.

BACKGROUND: Infections in hemodialysis (HD) patients lead to high morbidity and mortality rates and are associated with early cardiovascular mortality, possibly related to chronic inflammation. Intravenous (IV) iron is widely administered to HD patients and has been associated with increased oxidative stress and dysfunctional cellular immunity. The purpose of this study was to examine the effect of three commercially available IV iron preparations on intracellular reactive oxygen species generation and lymphocyte subpopulation survival. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from healthy donor buffy coat. PBMC were cultured and incubated with 100 microg/mL of sodium ferric gluconate (SFG), iron sucrose (IS) or iron dextran (ID) for 24 hours. Cells were then probed for reactive oxygen species (ROS) with dichlorofluorescein-diacetate. In separate studies, isolated PBMCs were incubated with the 25, 50 or 100 microg/mL iron concentrations for 72 hours and then stained with fluorescein conjugated monoclonal antibodies for lymphocyte subpopulation identification. Untreated PBMCs at 24 hours and 72 hours served as controls for each experiment. RESULTS: All three IV iron preparations induced time dependent increases in intracellular ROS with SFG and IS having a greater maximal effect than ID. The CD4+ lymphocytes were most affected by IV iron exposure, with statistically significant reduction in survival after incubation with all three doses (10, 25 and 100 microg/mL) of SFG, IS and ID. CONCLUSION: These data indicate IV iron products induce differential deleterious effects on CD4+ and CD16+ human lymphocytes cell populations that may be mediated by intracellular reactive oxygen species generation. Further studies are warranted to determine the potential clinical relevance of these findings.

Human papillomavirus type 16 E6 and E7 oncoproteins upregulate c-IAP2 gene expression and confer resistance to apoptosis.

Inhibition of apoptosis plays an important role in the cellular immortalization and transformation induced by E6 and E7 oncoproteins of human papillomavirus (HPV). Here, we report that the transcription of the inhibitor of apoptosis gene, cellular inhibitor of apoptosis protein 2, (c-IAP2), is significantly upregulated in HPV16 E6/E7-immortalized human oral keratinocytes (HOK16E6E7). Overexpression of E6/E7 from the high-risk HPV16 or 18, but not from the low-risk HPV6, activated c-IAP2 promoter. E6 from HPV16 and 18 played a major role in the activation. In addition, the induction of c-IAP2 transcription required nuclear factor-kappaB activity. Overexpression of c-IAP2 in normal human oral keratinocyte conferred resistance to tumor necrosis factor-alpha (TNF-alpha)/cycloheximide (CHX)-induced apoptosis, suggesting the increased c-IAP2 expression in HOK16E6E7 may protect the cells from TNF-alpha-mediated cell death. Moreover, depletion of endogenous c-IAP2 using RNA interference in HOK16E6E7 induced apoptosis, indicating that c-IAP2 is necessary for HPV16 E6/E7-induced resistance to apoptosis and cell survival. Of note, high levels of c-IAP2 transcription were found in several HPV16- or HPV18-positive cancer cells, and depletion of c-IAP2 caused cell death in HPV18-positive HeLa cells. Thus, upregulation of c-IAP2 by E6 and E7 may confer resistance to apoptosis that is necessary for sustained growth of some HPV16- and HPV18-positive cancer cells.

Non-association between acrochordons and colonic polyps in a minority population.

PURPOSE: To determine usefulness of skin tags as a predictor of colonic polyps, in patients of a minority population. SETTING: Inner-city community hospital serving predominantly African Americans and Hispanics. METHODS: Evaluation of 480 consecutive patients undergoing colonoscopy. The presence or absence of skin tags was noted, and their correlation with the colonic polyps determined. RESULTS: Colonic polyps were detected in 92 patients (19%). None of these patients had skin tags, whereas skin tags were found in 87 patients (18%), and none of them had colonic polyps. CONCLUSION: The mere presence of acrochordons (skin tags) should not be used as an indication for screening colonoscopy especially in African Americans and Hispanics.