Prognostic value of tumor deposits and positive lymph node ratio in stage III colorectal cancer: a retrospective cohort study.

BACKGROUND: In colorectal cancer (CRC), tumor deposits (TD) have been used to guide the N staging only in node-negative patients. It remains unknown about the prognostic value of TD in combination with positive lymph node ratio (LNR) in stage III CRC. PATIENTS AND METHODS: We analyzed data from 31,139 eligible patients diagnosed with stage III CRC, including 30,230 from the Surveillance, Epidemiology, and End Results (SEER) database as a training set and 909 from two Chinese hospitals as a validation set. The associations of TD and LNR with cancer-specific survival (CSS) and overall survival (OS) were evaluated using the Kaplan-Meier method and Cox regression models. RESULTS: Both TD-positive and high LNR (value≥0.4) were associated with worse CSS in the training (multivariable hazard ratio [HR], 1.50; 95% confidence interval [CI], 1.43-1.58 and HR, 1.74; 95% CI, 1.62-1.86, respectively) and validation sets (HR,1.90; 95%CI, 1.41-2.54 and HR,2.01; 95%CI, 1.29-3.15, respectively). Compared to patients with TD-negative and low LNR (value<0.4), those with TD-positive and high LNR had a 4.09-fold risk of CRC-specific death in the training set (HR, 4.09; 95% CI, 3.54-4.72) and 4.60-fold risk in the validation set (HR, 4.60; 95% CI, 2.88-7.35). Patients with TD-positive/H-LNR CRC on the right side had the worst prognosis (P<0.001). The combined variable of TD and LNR contributed the most to CSS prediction in the training (24.26%) and validation (32.31%) sets. A nomogram including TD and LNR showed satisfactory discriminative ability, and calibration curves indicated favorable consistency in both the training and validation sets. CONCLUSIONS: TD and LNR represent independent prognostic predictors for stage III CRC. A combination of TD and LNR could be used to identify those at high risk of CRC deaths.

Use of bioinformatic database analysis and specimen verification to identify novel biomarkers predicting gastric cancer metastasis.

Background: Gastric cancer (GC) is a common gastrointestinal tumor, and its metastasis has led to a significant increase in the death rate. The mechanisms of GC metastasis remain unclear. Methods: The differentially expressed genes (DmRs) and lncRNAs (DlncRs) of GC were selected from The Cancer Genome Atlas (TCGA) database. We applied the weighted gene co-expression network analysis (WGCNA) to construct co-expression modules related with GC metastasis. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) method analyzed the functional regions and signal pathways of genes in vital modules. DmRs-DlncRs co-expression network were drawn for finding out hub nodes. Survival analyses of significant biomarkers were analyzed by Kaplan-Meier (KM) method. Finally, the expressions of selected biomarkers were validated in cell lines and caner tissues by quantitative real-time PCR (qRT-PCR), in GC tissue microarray by Fluorescence in situ hybridization (FISH). Results: 4776 DmRs and 213 DlncRs were involved the construction of WGCNA network, and MEyellow module was identified to have more significant correlation with GC metastasis. DmRs and DlncRs of MEyellow module were proved to be involved in the processes of cancer pathogenesis by GO and KEGG pathway analysis. Through the DmRs-DlncRs co-expression network, 7 DmRs and 1 DlncRs were considered as hub nodes. Besides, the high expression of TIMD4, CETP, KRT27, PTGDS, FAM30A was worse than low expression in GC patients survival, respectively; However, LRRC26 was opposite trend. FAM30A and TIMD4 were all significant biomarkers of GC survival and hub genes. Simultaneously, TIMD4, CETP, KRT27, PTGDS, FAM30A were increased in GC cell lines and tissues compared with GES-1 and normal tissues, respectively; the expression of LRRC26 was reduced in GC cell lines and tissues. Conclusion: This study identified 6 genes as new biomarkers affecting the metastasis of GC. Especially, FAM30A and TIMD4 might be an effective marker for predicting the prognosis and a potential-therapeutic target in GC.

MAC30 Knockdown Inhibits Proliferation and Enhance Apoptosis of Gastric Cancer by Suppressing Wnt/ß-Cateninsignaling Pathway.

Gastric cancer is one of the most frequently diagnosed cancer and poses a serious threat to health system in the world. Upregulation of meningioma-associated protein (MAC30) has been found in many solid tumors and can regulate the proliferation, differentiation, and apoptosis of different tumor cells. Quantitative polymerase chain reaction (qPCR) was used to detect the expression of MAC30 in 68 patients with gastric cancer and their adjacent tissues. Lentiviral vector pGCSIL-shMAC30-GFP of the RNA interference (RNAi) of the MAC30 gene was transfected into gastric cancer BGC-823 cell line and the expression of lentivirus label protein GFP was observed via fluorescence microscope, while cell proliferation and apoptosis were determined with flow cytometry and MTT assay, respectively. Also, related protein expressions on Wnt/ß-catenin signaling pathway were analyzed by Western blot method. The expression of MAC30 was abnormally elevated in gastric cancer tissues, while interfering of its expression could significantly inhibit the proliferation of gastric cancer BGC-823 cell line. However, the promotion of apoptosis by mitochondrial pathway was mediated by Bax/Bcl-2 upregulation. Present work showed the effect of downregulated MAC30 expression on proliferation and apoptosis of gastric cancer cell through Wnt/ß-catenin signaling pathway. Thus, this investigation provides an experimental basis for future development of chemotherapeutic agent on gastric cancer.

4-1BB expression on MAIT cells is associated with enhanced IFN-γ production and depends on IL-2.

The role of MAIT cells in immunity against Mycobacterium tuberculosis infection in humans is still largely unexplored. In this study, we investigated the functional role of 4-1BB on MAIT cells. We found that 4-1BB was highly up-regulated on MAIT cells from tuberculous pleural effusions following Mtb antigen stimulation and its level of expression correlated with IFN-γ and IL-17 production. 4-1BB expression on MAIT cells in response to Mtb antigens was partially dependent on IL-2 and was associated with common γ chain receptor. By transcriptome sequencing, we identified numerous differentially expressed genes between 4-1BB- and 4-1BB+ MAIT cells. GO enrichment and KEGG pathway analysis of differentially expressed genes identified enriched pathways that included T-cell receptor and NF-κB signaling pathways. It is concluded that 4-1BB has the potential to be used as a biomarker to identify MAIT cells with enhanced IFN-γ and IL-17 responses that might be associated with tuberculosis infection control.

Synergistic efficacy of Cullin1 and MMP-2 expressions in diagnosis and prognosis of colorectal cancer.

BACKGROUND: Cullin1 and MMP-2 have been identified as important markers in various cancers, but their roles in colorectal cancer (CRC) have remained to be discovered. The aim of this study was to investigate the expression pattern and significance of Cullin1 and MMP-2 in CRC. METHODS: A total of 470 CRC patients were enrolled. Archival paraffin-embedded CRC tissue samples were used to generate tissue microarray blocks, which were immunohistochemically stained for Cullin1 and MMP-2. Prognostic and predictive role of Cullin1 and MMP-2 expression was evaluated by univariate and multivariate analysis, respectively. Values of p < 0.05 were considered statistically significant. RESULTS: Cullin1 and MMP-2 protein levels were significantly upregulated in CRC tissues compared with adjacent noncancerous tissues. High tumoral Cullin1 or MMP-2 expression significantly correlated with shorter overall survival (OS), as well as with clinicopathologic characteristics in patients. Multivariate regression analysis showed that high Cullin1 and MMP-2 expressions, separately and together, were independent negative markers of OS. CONCLUSION: Cullin1 and MMP-2 expressions could be novel diagnostic and prognostic markers for CRC patients.

Mucosal-associated invariant T cells from patients with tuberculosis exhibit impaired immune response.

OBJECTIVES: To identify factors which regulate MAIT cell response to Mycobacterium tuberculosis antigens, and to investigate the role of MAIT cells in patients with active tuberculosis. METHODS: Immune response of MAIT cells to M. tuberculosis antigens were compared between patients with active TB and healthy controls by flow cytometry and RNA sequencing. RESULTS: IFN-γ response of MAIT cells to M. tuberculosis lysates was dramatically improved by signal 3 cytokine IL-15 (p = 0.0002). Patients with active TB exhibited highly reduced IFN-γ production in MAIT cells stimulated with M. tuberculosis lysates/IL-15 compared with healthy controls (p < 0.0001) and individuals with latent TB infection (p = 0.0008). RNA sequencing of flow-sorted MAIT cells from patients with TB and healthy controls identified numerous differentially expressed genes, and the expression of genes that encode IFN-γ, TNF-α, IL-17F, granulysin and granzyme B were all down-regulated in patients with TB. MAIT cells from patients with TB has significantly lower expression of γc receptor than those from healthy controls under condition of Mtb lysates/IL-15 stimulation (p = 0.0028). Blockade of both γc and IL-2Rß receptors resulted in highly reduced frequency of IFN-γ-producing MAIT cells (79.4%) (p = 0.0011). CONCLUSIONS: MAIT cells from patients with active TB exhibited impaired cytokine and cytotoxic response to M. tuberculosis antigens.

Ternary Dinuclear Copper(II) Complexes of a Reduced Schiff Base Ligand with Diimine Coligands: DNA Binding, Cytotoxic Cell Apoptosis, and Apoptotic Mechanism.

A serial of mixed-ligand Cu(II) complexes of the type [Cu(phens)(H2 PDILeu)]H2 O (1-4) containing phens as 2,2'-bipyridyl (bpy, 1), 1,10-phenanthroline (phen, 2), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 3), and dipyrido[3,2-a:2',3'-c]phenazine (dppz, 4) have been isolated and characterized. The interaction of the complexes with calf-thymus DNA has been explored by physical methods to propose modes of DNA binding of the complexes, which indicate that 4 interacts with DNA more strongly than all of the other complexes through intercalation interaction. Furthermore, cell apoptosis was detected by AnnexinV/PI flow cytometry and TUNEL assay and by Western blotting to detect the protein expression of p53, Bax, and Bcl-2. All the three copper complexes can effectively induce apoptosis of the three human tumor cells, which was accompanied with upregulation of the expression of p53 and Bax, while Bcl-2 decreased.

Elevated expression of T-bet in mycobacterial antigen-specific CD4(+) T cells from patients with tuberculosis.

T-bet is a T-box transcriptional factor that controls the differentiation and effector functions of CD4 T cells. In this study, we studied the role of T-bet in regulating CD4(+) T cell immunity against tuberculosis (TB). T-bet expression in Mycobacterium tuberculosis antigen-specific CD4(+) T cells was significantly higher in patients with active TB than in individuals with latent TB infection (p<0.0001). Comparison of T-bet expression in TCM and TEM subsets showed that CD4(+)T-bet(+)M. tuberculosis antigen-specific CD4(+) T cells had significantly lower frequency of TCM (p=0.003) and higher frequency of TEM (p=0.003) than CD4(+)T-bet(-) cells. The expression of PD-1 in antigen-specific CD4(+) T cells was significantly higher in patients with TB than in individuals with latent TB infection (p=0.006). CD4(+)CD154(+)T-bet(+) T cells had significantly higher expression of PD-1 than CD4(+)CD154(+)T-bet(-) T cells (p=0.0028). It is concluded that T-bet expression might be associated with differentiation into effector memory cells and PD-1 expression in mycobacterial antigen-specific CD4(+) T cells.

miR-223 is upregulated in monocytes from patients with tuberculosis and regulates function of monocyte-derived macrophages.

Tuberculosis (TB) is a serious infectious disease that most commonly affects the lungs. Macrophages are among the first line defenders against establishment of Mycobacterium tuberculosis infection in the lungs. In this study, we found that activation and cytokine production in monocyte-derived macrophages (MDM) from patients with active TB was impaired. miR-223 expression was significantly elevated in monocytes and MDM from patients with TB compared with healthy controls. To determine the functional role of miR-223 in macrophages, stable miR-223-expressing and miR-223 antisense-expressing U937 cells were established. Compared with empty vector controls, expression of IL-1ß, IL-6, TNF-α and IL-12p40 genes was significantly higher in miR-223 antisense-expressing U937 cells, but lower in miR-223-expressing U937 cells. miR-223 can negatively regulate activation of NF-κB by inhibition of p65 phosphorylation and nuclear translocation. It is concluded that miR-223 can regulate macrophage function by inhibition of cytokine production and NF-κB activation.

Erythropoietin (EPO) protects against high glucose-induced apoptosis in retinal ganglional cells.

The aim of this study was to investigate the protective effect and mechanism of EPO on the apoptosis induced by high levels of glucose in retinal ganglial cells (RGCs). High glucose-induced apoptosis model was established in RGCs isolated from SD rats (1-3 days old) and identified with Thy1.1 mAb and MAP-2 pAb. The apoptosis was determined by Hochest assay. The levels of ROS were quantitated by staining the cells with dichloro-dihydro-fluorescein diacetate (DCFH-DA) and measure by flow cytometry. The SOD, GSH-Px, CAT activities, and levels of T-AOC and MDA were determined by ELISA. Change in mitochondrial membrane potential (Δψm) was also assessed by flow cytometry, and expressions of Bcl-2, Bax, caspase-3, caspase-9, and cytochrome C were assessed by western blotting. The RGCs treated with high glucose levels exhibited significantly increased apoptotic rate and concentrations of ROS and MDA. Pretreatment of the cells with EPO caused a significant blockade of the high glucose-induced increase in ROS and MDA levels and apoptotic rate. EPO also increased the activities of SOD, GSH-Px, and CAT, and recovered the levels of T-AOC levels. As a consequence, the mitochondrial membrane potential was improved and Cyt c release into the cytoplasm was prevented which led to significantly suppressed up-regulation of Bax reducing the Bax/Bcl-2 ratio. The expressions of caspase-3 and caspase-9 induced by high glucose exposure were also ameliorated in the RGCs treated with EPO. The protective effect of EPO against apoptosis was mediated through its antioxidant action. Thus, it blocked the generation of pro-apoptotic proteins and apoptotic degeneration of the RGCs by preventing the mitochondrial damage.

Mucosal-associated invariant T-cell function is modulated by programmed death-1 signaling in patients with active tuberculosis.

RATIONALE: Mucosal-associated invariant T (MAIT) cells have been proven to play an important role in host defense against mycobacterial infection in animal infection models; however, the functional role of MAIT cells in patients with active tuberculosis (TB) is still largely unknown. OBJECTIVES: To understand the clinical features and functions of MAIT cells in patients with active TB. METHODS: MAIT cells were analyzed in patients with pulmonary TB, tuberculous pleurisy, and tuberculous peritonitis by flow cytometry. The functions of MAIT cells were compared between patients with active TB and healthy control subjects. MEASUREMENTS AND MAIN RESULTS: The frequency of MAIT cells was significantly reduced both in peripheral blood from patients with active pulmonary TB (P < 0.0001) and in tuberculous pleural effusions compared with healthy control subjects but not in ascitic fluids from patients with tuberculous peritonitis. A comparison of bacillus Calmette-Guérin (BCG)-stimulated cytokine production showed that patients with active TB had significantly higher production of IFN-γ (P = 0.0034) and tumor necrosis factor (TNF)-α (P = 0.0399) compared with healthy control subjects. In contrast, when MAIT cells were stimulated with Escherichia coli, patients with active TB had significantly lower production of IFN-γ (P = 0.0007) and TNF-α (P = 0.0032). MAIT cells in patients with active TB exhibited elevated expression of programmed death-1 (PD-1) (P = 0.0015), and blockade of PD-1 signaling resulted in a significantly higher frequency of BCG-stimulated IFN-γ production in MAIT cells (P = 0.0178). CONCLUSIONS: MAIT-cell immune response to antigen stimulation in patients with active TB is regulated by PD-1, which could be a potential target for TB immunotherapy.

An anti-human CD13 monoclonal antibody that suppresses the suppressive function of Treg cells.

CD13 (CD13/aminopeptidase N, APN, or CD13/APN) is a widely expressed type II membrane-bound metalloprotease. It is often overexpressed on cancer cells and expressed on CD4(+)CD25(hi) Treg cell subpopulation with higher suppressive ability. It has been determined to be a promising target in cancer diagnosis and therapy. In this study, a functional anti-human CD13 monoclonal antibody, MAb 9E4, was obtained and the specificity of this MAb was verified by flow cytometry. This MAb effectively recognized the CD13 molecule expressed on a series of malignant cell lines. Furthermore, we demonstrated that MAb 9E4 suppresses the suppressive function of Treg cells. This functional anti-human CD13 MAb provides a valuable tool for further study targeting the CD13 positive Treg cells.

Type II, but not type I, cGMP-dependent protein kinase reverses bFGF-induced proliferation and migration of U251 human glioma cells.

Previous data have shown that the type II cGMP­dependent protein kinase (PKG II) inhibits the EGF­induced MAPK signaling pathway. In order to thoroughly investigate PKG, it is necessary to elucidate the function of another type of PKG, PKG I. The aim of this study was to investigate the possible inhibitory effect of PKG II and PKG I activity on the basic fibroblast growth factor (bFGF)­induced proliferation and migration of U251 human glioma cells and the possible underlying mechanisms. U251 cells were infected with adenoviral constructs encoding cDNA of PKG I (Ad­PKG I) or PKG II (Ad­PKG II) to increase the expression levels of PKG I or PKG II and then treated with 8­Br­cGMP and 8­pCPT­cGMP, respectively, to activate the enzyme. An MTT assay was used to detect the proliferation of the U251 cells. The migration of the U251 cells was analyzed using a Transwell migration assay. Western blot analysis was used to detect the phosphorylation/activation of the fibroblast growth factor receptor (FGFR), MEK and ERK and the nuclear distribution of p-ERK. The results showed that bFGF treatment increased the proliferation and migration of U251 cells, accompanied by increased phosphorylation of FGFR, MEK and ERK. Furthermore, the nuclear distribution of p-ERK increased following bFGF treatment. Increasing the activity of PKG II through infection with Ad-PKG II and stimulation with 8-pCPT-cGMP significantly attenuated the aforementioned effects of the bFGF treatment, while increased PKG I activity did not inhibit the effects of bFGF treatment. These data suggest that increased PKG II activity attenuates bFGF­induced proliferation and migration by inhibiting the MAPK/ERK signaling pathway, whereas PKG I does not.

Endogenous cGMP-dependent protein kinase reverses EGF-induced MAPK/ERK signal transduction through phosphorylation of VASP at Ser239.

In our previous study, we demonstrated that type II cGMP-dependent protein kinase (PKG II) was expressed at lower levels in different human cancer cell lines and that exogenous PKG II inhibited epidermal growth factor (EGF)-induced MAPK/ERK signaling. In order to investigate its functions further in this signaling pathway, it is necessary to elucidate whether endogenous PKG has the same effect or not. This study aimed to investigate the possible inhibitory effect of endogenous PKG activity on EGF-induced MAPK/ERK signal transduction in human lung cancer cells and its mechanism. Human small cell lung carcinoma cells (SCLCs) were treated with the PKG-selective cGMP analog 8-pCPT-cGMP to activate endogenous PKG, EGF and cGMP followed by EGF, respectively. The results showed that increased endogenous PKG activity inhibited the EGF-induced phosphorylation of the epidermal growth factor receptor (EGFR) and the binding between Sos1 and Grb2. In addition, EGF-triggered Ras activation was reversed by increased endogenous PKG activity. While the EGF-induced phosphorylation of MEK and ERK were inhibited by increased endogenous PKG activity, there was a significant increase of phosphorylated vasodilator-stimulated phosphoprotein (p-VASP) at Ser239. Furthermore, we investigated whether endogenous PKG exerted its effects on EGF-induced MAPK/ERK signaling through phosphorylation of VASP at Ser239. Downregulation of the levels of p-VASP Ser239 by point mutation blocked the effects of endogenous PKG on EGF-induced MAPK/ERK signal transduction. The data shown here suggest that endogenous PKG reverses the EGF-induced MAPK/ERK signaling pathway by phosphorylating VASP at Ser239.

Diagnostic performance of multiplex cytokine and chemokine assay for tuberculosis.

Simultaneous detection of multiple biomarkers might lead to improved diagnostic performance for Mycobacterium tuberculosis infection. In this study, we screened soluble biomarkers that had significant differences in patients with active tuberculosis and healthy controls and evaluated the diagnostic performance of the multiplex cytokine/chemokine assay. Overall, 178 patients with active pulmonary tuberculosis, 156 healthy individuals and 35 patients with bacterial pneumonia or lung cancer were evaluated. Among the 16 soluble biomarkers screened by the microbead-based multiplex assay, five cytokines/chemokines including IFN-γ, IP-10, MIG, TNF-α and IL-2 that showed most significant differences between active pulmonary tuberculosis patients and healthy controls were selected for further analysis. When analyzed individually, both IP-10 and MIG had sensitivity and specificity comparable to IFN-γ in detection of active TB. Combined detection of IFN-γ, IP-10 and MIG had significantly improved sensitivity and specificity as compared with individual cytokine and chemokine detection. The responsive levels of IFN-γ, IP-10, MIG, TNF-α and IL-2 were significantly lower in re-treatment pulmonary tuberculosis patients than in new tuberculosis patients. It is concluded that combined IFN-γ, IP-10, MIG multiplex detection had better diagnostic performance for tuberculosis than the individual cytokine/chemokine assays. The re-treatment pulmonary tuberculosis patients had poor responses to ESAT-6/CFP-10 peptides stimulation.

Reduced CD27 expression on antigen-specific CD4+ T cells correlates with persistent active tuberculosis.

OBJECTIVE: CD27, a member of the tumor necrosis factor receptor family, has important role in generation of T cell immunity. In this study, association of CD27 expression on mycobacterial antigen-specific CD4+ T cells with pulmonary tuberculosis (TB) was investigated. METHODS: Mycobacterial antigen-specific CD4+ T cells were identified based on CD154 expression and CD27 expression on antigen-specific CD4 T cells was analyzed by flow cytometry. RESULTS: Compared with tuberculin-positive controls, patients with bacterial culture-positive pulmonary TB had significantly reduced CD27 expression on antigen-specific CD4 T cells. The persistent active TB patients had much lower percentages of CD27+ antigen-specific CD4 T cells than culture-positive new TB patients (P=0.008) and healthy controls (P=0.005). Logistic regression analysis on frequencies of CD27-expressing antigen-specific CD4 T cells and TB patients' clinical characteristics indicated that low percentage of CD27+ antigen-specific CD4 T cells correlated significantly with persistent active tuberculosis (P=0.002, odds ratio=19.6). CONCLUSION: It is concluded that frequency of CD27+ antigen-specific CD4 T cells could be used as an immunological marker for persistent active TB.

Association of mycobacterial antigen-specific CD4(+) memory T cell subsets with outcome of pulmonary tuberculosis.

OBJECTIVES: Memory T cells are hallmark of acquired immunological responses. The relationship of mycobacterial antigen-specific CD4(+) memory T cell subsets with pulmonary tuberculosis was investigated. METHODS: The mycobacterial antigen-specific CD4(+) T cells were detected based on CD154 expression and phenotypes of memory T cell were analyzed by surface staining of CD45RA and CCR7 and flow cytometrical analysis in patients with pulmonary tuberculosis and in tuberculin-positive healthy controls. The association of antigen-specific CD4(+) memory T cell subsets with disease severity and anti-TB treatment was analyzed in patients with pulmonary tuberculosis. RESULTS: Patients with pulmonary tuberculosis had significantly lower frequencies of antigen-specific central memory T cells (T(CM)) (p=0.019) and higher frequencies of effector memory T cells (T(EM)) (p=0.022) compared with tuberculin-positive healthy controls without tuberculosis. Patients with smear/culture positive results showed lower population frequencies of T(CM) and significantly higher frequencies of T(EM) (p=0.015) than those with smear/culture negative results. Treatment of TB patients with standard antibiotic regimens for more than one month led to significantly increased frequencies of T(CM) (p=0.031). CONCLUSIONS: The frequencies of mycobacterial antigen-specific T(CM) and T(EM) are associated with disease severity of pulmonary tuberculosis and T(CM) are associated with short-term effects of anti-TB chemotherapy.

Sulfur (S)-induced enhancement of iron plaque formation in the rhizosphere reduces arsenic accumulation in rice (Oryza sativa L.) seedlings.

The effects of two sulfur (S) sources (SO(4)(2-), S(0)), and three rates of S application (0, 30, 120 mgS/kg) on the formation of iron plaque in the rhizosphere, and on the root surface of rice, and As (arsenic) uptake into rice (Oryza sativa L.) were studied in a combined soil-sand culture experiment. Significant differences in As uptake into rice between +S and -S treatments were observed in relation to S sources, and rates of S application. Concentrations of As in rice shoots decreased with increasing rates of S application. The mechanism could be ascribed to sulfur, induced the formation of iron plaque, since concentrations of Fe in iron plaque on quartz sands in the rhizosphere, and on the root surface of rice increased with increasing rates of S application. The results suggest that sulfur fertilization may be important for the development approaches to reducing As accumulation in rice.

Enhanced therapeutic efficacy for ovarian cancer with a serotype 3 receptor-targeted oncolytic adenovirus.

Oncolytic viruses that are replication competent in tumor but not in normal cells represent a novel approach for treating neoplastic diseases. However, the oncolytic potency of replicating agents is determined directly by their capability of infecting target cells. Most adenoviruses used for gene therapy or virotherapy have been based on serotype 5 (Ad5). Unfortunately, expression of the primary receptor for Ad5 (the coxsackie-adenovirus receptor, or CAR) is highly variable on ovarian and other cancer cells. By performing genetic fiber pseudotyping, we created Ad5/3-Delta24, a conditionally replicating adenovirus that does not bind CAR but facilitates entry into and killing of ovarian cancer cells. We show replication of Ad5/3-Delta24 and subsequent oncolysis of ovarian adenocarcinoma lines. Replication was also analyzed with quantitative PCR on three-dimensional primary tumor cell spheroids purified from patient samples. Moreover, in a therapeutic orthotopic model of peritoneal carcinomatosis, dramatically enhanced survival was noted. Finally, Ad5/3-Delta24 achieved a significant antitumor effect as assessed by noninvasive, in vivo bioluminescence imaging. Therefore, the preclinical therapeutic efficacy of Ad5/3-Delta24 is improved over the respective CAR- and integrin-binding controls. Taken together with promising biodistribution and toxicity data, this approach could translate into successful clinical interventions for ovarian cancer patients.

Blood-based screening and light based imaging for the early detection and monitoring of ovarian cancer xenografts.

We report a novel technology for in vivo early detection, identification, and monitoring of ovarian cancer in live mice leading to better treatment outcome. A genetic dualistic reporter system that uses an adenoviral (Ad) vector to transfer the genetic reporters to the ovarian cancer is described. Infection of the cancer cells leads to expression of one reporter that is detected in blood, namely, secreted human placental alkaline phosphatase (SEAP). A second reporter, namely, enhanced green fluorescent protein (GFP) is also delivered by the Ad, leading to expression at the site of ovarian cancer. The SEAP gene under control of the cytomegalovirus (CMV) promoter element is linked to the GFP gene with an IRES element. A diagnostic adenoviral vector (Ad) encoding the SEAP and GFP (Ad5-SEAP-GFP) is produced. Efficacy of newly developed diagnostic vector is tested in cell culture and animal models. SKOV3ip.1 cells are infected with Ad5-SEAP-GFP. Over time the cells are monitored for fluorescence and SEAP is also measured in the growth media supernatant. For animal experiments, SKOV3ip.1 cells are implanted first in nude mice either subcutaneously (SC) or intraperitoneally (IP) separately. After 4-7 days, the Ad5-SEAP-GFP is administered. Control mice do not receive any Ad vector. All mice are imaged with a fluorescent stereomicroscope after 24 h, and blood is collected for SEAP analyses. Increasing green fluorescence is detected in all SKOV3ip.1 cells infected with Ad5-SEAP-GFP, while SEAP levels in growth media increase over monitoring period. Expression of GFP in both SC and IP tumors is detected by 24 h in the live mice. At this time, the SEAP blood levels are more than 2-3 fold greater than blood levels of control group. GFP fluorescence and SEAP levels continue to increase in all mice that are injected with Ad5-SEAP-GFP until termination. Control mice (both SC and IP) do not express GFP or SEAP throughout the experiment. GFP contrast is necessary to differentiate between micro-sized early stage non-palpable ovarian tumor nodules and surrounding normal tissue. While the studies are conducted in mice, it is envisioned that the dual-based approach will eventually be translated into human applications for routine diagnosis and monitoring of ovarian cancer when an ovarian cancer specific promoter will be available. Due to the thickness of the abdominal wall in human laparoscopy or laparotomy will be necessary. This system will provide gynecologic oncologists with a more effective tool for treating patients. The blood-based screening assay provides a quick test to determine the presence of the ovarian cancer at its earliest stage. The location of the ovarian cancer is afforded by the light-based imaging component, which represents a new and improving technology with tremendous advantages of sensitivity and spatial resolution to localize micro-sized tumor nodules. The novelty of the dualistic system is the linkage of blood-based reporter screening as a selection criteria for subsequent light-based imaging procedures. This combination will lead to an accurate and widely applicable method for the early detection and monitoring of ovarian cancer, especially in high-risk women