TNF-alpha mRNA Research Articles

RNA Interference Targeting p110β Reduces Tumor Necrosis Factor-Alpha Production in Cellular Response to Wear Particles In vitro and Osteolysis In vivo

Aseptic joint loosening is a key factor that reduces the life span of arthroplasty. There are currently few effective treatments for joint loosening except surgical revision. We explored the inhibitory effects of p110β-targeted small interfering RNA (siRNA) and lentivirus on particle-induced inflammatory cytokine expression in the murine macrophage cell line, RAW264.7. siRNA- and lentivirus-targeting p110β were transfected and infected prior to particle stimulation, respectively. Ceramic and titanium particles of different sizes were prepared to stimulate macrophages. Fluorescence microscopy showed that the efficiency of siRNA transfection and lentivirus infection were 74.2 ± 4.2 and 92.3 ± 2.6 %, respectively. TNF-alpha mRNA in the particle stimulation plus RNA interference (RNAi) groups were significantly lower compared with the particle stimulation-only groups (P < 0.05). Similarly, protein levels of TNF-alpha in RNAi-treated groups were significantly decreased after transfection or infection (P < 0.05). It showed that Phosphor-AKT (Ser473) activation was significantly reduced by RNAi through western blot. As assessed by CT, micro-CT and histological analysis, particle implantation induced a significant osteolysis in mice calvaria, which was limited by p110β lentivirus addition. These results suggested that p110β subtype of PI3K, followed by activation of Ser473, may possibly participate in the regulation of macrophages activity by wear particles, ultimately resulting in the TNF-α secretion and osteolysis.

Effects of salidroside pretreatment on expression of tumor necrosis factor-alpha and permeability of blood brain barrier in rat model of focal cerebralischemia-reperfusion injury

ObjectiveTo observe changes in expression of tumor necrosis factor (TNF)-alpha and permeability of blood brain barrier after salidroside pretreatment in rats with injury induced by focal cerebralischemia-reperfusion. MethodsForty-five male SD rats were randomly divided into three groups (n=15): control group, ischemia-reperfusion (IR) model group, and salidroside pretreatment group. Before the IR model establishment, the rats in the salidroside pretreatment group were intraperitoneally administered with salidroside at a dose of 24 mg/(kg·d) for 7 d. After 30 min post the last administration, the IR model was induced by occlusion of middle cerebral artery with a filament. After 24 h post the operation, the water content and Evens blue content in the ischemia cerebral hemisphere were determined, and the level of TNF-alpha mRNA was detected by the semi-quantitative RT-PCR. ResultsCompared with the IR model group, the salidroside pretreatment group had significantly lower (P<0.05) water content and Evens blue content in the ischemia cerebral hemisphere and also had significantly lower (P<0.05) level of TNF-alpha in the ischemic cerebral cortex tissue. ConclusionsThe salidroside pretreatment alleviated the focal cerebralischemia-reperfusion injury in the rat model, possibly by decreasing the permeability of blood brain barrier, attenuating brain edema and reducing TNF-alpha expression.

Accelerated fat cell aging links oxidative stress and insulin resistance in adipocytes

Telomere shortening is emerging as a biological indicator of accelerated aging and aging-related diseases including type 2 diabetes. While telomere length measurements were largely done in white blood cells, there is lack of studies on telomere length in relation to oxidative stress in target tissues affected in diabetes. Therefore, the aim of this study is to induct oxidative stress in adipocytes and to test whether these adipocytes exhibit shortened telomeres, senescence and functional impairment. 3T3-L1 adipocytes were subjected to oxidative stress and senescence induction by a variety of means for 2 weeks (exogenous application of H2O2, glucose oxidase, asymmetric dimethylarginine (ADMA) and glucose oscillations). Cells were probed for reactive oxygen species generation (ROS), DNA damage, mRNA and protein expression of senescent and pro-inflammatory markers, telomere length and glucose uptake. Compared to untreated cells, both ROS generation and DNA damage were significantly higher in cells subjected to oxidative stress and senescence. Adipocytes subjected to oxidative stress also showed shortened telomeres and increased mRNA and protein expression of p53, p21, TNF alpha and IL-6. Senescent cells were also characterized by decreased levels of adiponectin and impaired glucose uptake. Briefly, adipocytes under oxidative stress exhibited increased ROS generation, DNA damage, shortened telomeres and switched to senescent/pro-inflammatory phenotype with impaired glucose uptake.

A new feature of importance for the TNF-alpha system in inflammation—Bilateral myositis that develops early in response to unilateral overuse shows a marked involvement of TNF-alpha not only in the exercised side but also contralaterally

Using a rabbit model leading to myositis in response to exercise-induced muscle overuse, we have previously observed that TNF-alpha is involved in the exercised muscle in early developing myositis as well as both ipsiand contralaterally in the myositis which develops in response to a lengthened period of overuse. It is unknown if TNF-alpha can also be engaged contralaterally in early stages of myositis. The hypothesis was that this is the case. It was therefore evaluated whether the TNF-alpha system is early involved contralaterally. An experimental model of 1 week of overuse of the soleus muscle on one side leading to myositis was used, and in situ hybridization and immunohistochemistry were applied to study the expression patterns of TNF-alpha in the soleus muscle in the contralateral side. TNF-alpha was expressed in the myositis process which occurred contralaterally. There were thus TNF-alpha mRNA reactions in the cells of the inflammatory infiltrates, in blood vessel walls and in certain of the muscle fibers. Parts of the latter were necrotic fibers, whereas others were interpreted to be in a regenerative stage. TNF-alpha immunoreactions were seen for infiltrating white blood cells. The observations show that the TNF-alpha system is early involved in the cross-over effects that occur in response to unilateral muscle overuse leading to myositis bilaterally. TNF-alpha is likely to have pro-inflammatory and destructive effects but also to have effects in the muscle regenerative processes. The occurrence of an early involvement of the TNF-alpha system contralaterally to the injury side shows a new aspect of importance of this system in inflammation.

Equine colostral carbohydrates reduce lipopolysaccharide‐induced inflammatory responses in equine peripheral blood mononuclear cells

Increasing evidence suggests that reactions to lipopolysaccharide (LPS), particularly in the gut, can be partly or completely mitigated by colostrum- and milk-derived oligosaccharides. Confirmation of this hypothesis could lead to the development of new therapeutic concepts. To demonstrate the influence of equine colostral carbohydrates on the inflammatory response in an in vitro model with equine peripheral blood mononuclear cells (PBMCs). Carbohydrates were extracted from mare colostrum, and then evaluated for their influence on LPS-induced inflammatory responses in PBMCs isolated from the same mares, mRNA expression of tumour necrosis factor-alpha, interleukin-6 and interleukin-10 was measured as well as the protein levels of tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10). Equine colostral carbohydrates significantly reduced LPS-induced TNF-alpha protein at both times measured and significantly reduced LPS-induced TNF-alpha, IL-6 and IL-10 mRNA expression by PBMCs. Moreover, cell viability significantly increased in the presence of high concentrations of colostral carbohydrates. Carbohydrates derived from equine colostrum reduce LPS-induced inflammatory responses of equine PBMCs. Colostrum and milk-derived carbohydrates are promising candidates for new concepts in preventive and regenerative medicine.

Abstract 18445: Potential Contribution of RANKL System in Adipose Tissue Inflammation and Insulin Resistance

Hypertension and obesity are related to adipose tissue inflammation and insulin resistance, and both are important factors for the development of type II Diabetes mellitus. High serum osteoprotegerin (OPG) level has been reported in diabetic patients, and is considered a marker for future cardiovascular events. OPG is a decoy receptor of RANKL, and inhibits its binding to the receptor RANK. The RANKL system has been reported in bone, mammary gland development, immune cells communication, and we have previously reported its role in vascular calcification and potential cross-talk with Renin-angiotensin II system via ERK phosphorylation pathway. In this study, we examined the role of RANKL system in adipose tissue. High fat diet significantly increased the RANK-RANKL, ACE, AT1R as well as TNF alpha and MCP-1 mRNA expression in C57/BL6 and db/db mice adipose tissue. In vitro studies showed that RANKL expression increases following human pre adipocyte differentiation, and is further increased in the presence of Angiotensin II. We have found that PMA-activated monocytes (THP1) express higher level of RANK compared to non-stimulated monocytes, and that RANKL stimulation induces inflammatory cytokines expression in the presence of anti-OPG neutralizing antibody. Also, RANKL induced macrophage migration in boyden chamber assay dose dependently. OPG-/- mice under high fat diet presented increased number of crown-like structures in adipose tissue compared to wild type. Interestingly the deletion of AT1R in OPG-/-mice significantly decreased that macrophage infiltration, and inhibited the expression of inflammatory cytokines and RANKL system in adipose tissue. OPG-/- mice, known as the model of continuous activation of RANKL system, showed higher insulin level and insulin resistance compared to WT and AT1R-/-/OPG-/- mice. Overall, we propose that RANKL is activated in adipose tissue under high fat diet, and may contribute to insulin resistance by inducing macrophage recruitment and adipose tissue inflammation. Moreover, blockade of local renin-angiotensin II activation can also inhibit RANKL system activation in adipose tissue.

Effect of sophoridine and TLR4/MD-2 blocking agent on pathway of LPS-induced RAW264.7 macrophage TLR4-NF-κB-TNF-α

To study the effect of sophoridine and TLR4/MD-2 blocking agent on pathway of LPS-induced RAW264. 7 macrophage TLR4-NF-kappaB-TNF-alpha in and its pharmacological mechanism of antiendotoxin. RAW264. 7 macrophages were cultured and divided into 5 groups, namely the blank control group, the LPS model group, the sophoridine + LPS group, the TLR4/MD-2 blocking agent + LPS group and the anti-TLR4/MD-2 + sophoridine + LPS group. Cells and cell culture fluids were collected at 120 min after the each group was processed. The expression of TLR4 protein was measured by western blot, the distribution and expression of NF-kappaB protein were measured by immunocytochemistry, and the expression of NF-kappaB and TNF-alpha mRNA were measured by western blot and reverse transcriptase polymerase chain reaction (RT-PCR). The content of TNF-alpha in the cell supernatant was detected by using radioimmunoassay. Compared with the LPS group, the expression of TLR4 protein, NF-kappaB mRNA, the rate of NF-kappaB entry the nucleus, TNF-alpha mRNA and TNF-alpha content in the cell supernatant were significantly decreased in the sophoridine + LPS group (P < 0.01). The rate of NF-kappaB entry the nucleus and TNF-alpha in the TLR4/MD-2 blocking agent + LPS group and the TLR4/MD-2 blocking agent + sophoridine + LPS group were notablly lower than that of the LPS model group (P < 0.01), close to that of the blank control group. However, there was no statistical significance between the two groups. TLR4/MD-2 may be one of sophoridine's targets. Sophordine's inhibitory effect on pathway activity of TLR4-NF-kappaB-TNF-alpha may be one of its antiendotoxin mechanisms.

Exogenous Ghrelin Administration Can Ameliorate Indomethacin-Induced Small Intestinal Damage in Mice

Purpose: The arrival of video capsule endoscopy and balloon enteroscopy revealed that non-steroidal anti-inflammatory drugs (NSAIDs) can cause more frequent and more serious complications than formerly believed in the small intestine. However, safe and effective treatment is not available for NSAIDs-induced small intestinal damage. As a possible candidate, we focused on ghrelin, an orexigenic peptide secreted mainly from stomach. Although previous reports revealed that this peptide might show gastroprotection, the effect of ghrelin on NSAIDs-induced small intestinal damage was still vague. In order to examine its therapeutic potential for NSAIDs-induced small intestinal damage, we analyzed the influence of exogenous ghrelin administration on small intestinal protection using experimental mouse model. Methods: Indomethacin or vehicle was subcutaneously administrated to C57BL/6J mice, and ghrelin or vehicle was intraperitoneally administrated before 30 minutes and 2 hours afterwards (vehicle/vehicle group, vehicle/ghrelin group, indomethacin/vehicle group, and indomethacin/ghrelin group). Small intestinal injuries were assessed by macroscopical and microscopical alterations, myeloperoxidase (MPO) activity, prostaglandin E2 (PG E2) and expression of mRNA for cox-1, cox-2, iNOS, TNF-alpha, IL-1beta, and IL-6. Results: Vehicle/vehicle group and vehicle/ghrelin group did not show small intestinal alterations. Indomethacin/ghrelin group significantly modified macroscopical and microscopical scores, and also attenuated NSAIDs-induced changes in MPO activity, PG E2 level and mRNA expression compared those in indomethacin/vehicle group. Conclusion: Ghrelin shows a strong protective efficacy against indomethacin-induced small intestinal damage in mice. Also considering the previous reports on gastroprotective role, ghrelin can prevent NSAIDs-induced mucosal damage through the whole digestive tract, which will lead to a new treatment approach toward the increase of ghrelin expression.

OMC-2010 추출물이 마우스의 비장세포 cytokine 생성에 미치는 영향

Objective : This study was performed to estimate the effects of OMC-2010 extract on cytokine production in mouse spleen cells. Methods : Mouse spleen cells were pre-treated with ethanol and water extract of OMC-2010 for 1 h, then stimulated with lipopolysaccharide (LPS, 1 <TEX>${\mu}g/ml$</TEX>) for 48 h. Then the cells were harvested for real-time reverse transcription polymerase chain reaction to detect cytokines. Results : OMC-2010 ethanol extract significantly inhibited the LPS-induced interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and IL-5 mRNA expressions, but not shown such changes in IL-6, IL-4, IL-13. OMC-2010 water extract significantly inhibited the LPS-induced TNF-alpha, and IL-5 mRNA expressions, but not shown such changes in IL-1beta, IL-6, IL-4, IL-13. Conclusions : Theses results could suggest that both ethanol and water OMC-2010 extract could inhibit the TNF-alpha and IL-5 mRNA expression.

P079 ARID5a is an IL-6 mRNA stability protein. Chlorpromazine mediates its inhibitory effect on IL-6 production in macrophages through inhibition of ARID5A expression

Introduction Our previous study showed that chlorpromazine (CPZ), an anti-histamine therapeutic, inhibits LPS-induced IL-6 but not TNF-α production in macrophages [1] . In the current study, we show that CPZ is an inhibitor of LPS-induced IL-6 but not TNF-α mRNA stability. Since the 3′-untranslated region (3′-UTR) is important for the regulation of IL-6 mRNA stability, we examined whether CPZ affects the activity of RNA stabilizing proteins on AU-rich IL-6 3′-UTR mRNA. Here we identified a novel IL-6 mRNA stability protein, Arid5a. The Arid (AT-rich interactive domain) family of proteins have been previously demonstrated to be involved in the control of differentiation, tissue-specific gene expression and proliferation control [2] . In particular Arid5a was shown to be involved in chondrocyte-specific gene transcription [3] . In this study, we report a novel function for Arid5a on the stabilization of IL-6 mRNA. Methods For knockdown of Arid5a gene expression we employed siRNA technology. Levels of IL-6 and TNF-alpha protein in RAW 264 macrophage cell supernatants following transfection of siRNA control or ARID5a, were determined by ELISA. IL-6 and TNF-alpha mRNA stability was determined by real-time PCR. Interaction of ARID5a with the IL-6 3′-UTR was examined by mass spectrometry (LC-MS/MS). Results We found that Arid5a is induced by LPS stimulation in macrophages and binds to the IL-6 3′-UTR in macrophages. In RAW 264 cells, knockdown of Arid5a by siRNA led to the decrease of LPS-induced IL-6 but not TNF-α production. We showed that overexpression of Arid5a critically stabilizes IL-6 mRNA through AU-rich elements of the 3′-UTR region. Conclusion Thus, Arid5a is an IL-6 mRNA stability protein which mediates the inhibitory effects of CPZ on LPS-mediated IL-6 production. CPZ, or novel derivatives of this molecule, may represent a new therapy for IL6-dependent diseases (such as arthritis), through its inhibitory function on IL-6 mRNA stability.

Regulating effect of Ginkgo biloba extract 50 on hippocampal inflammation-related cytokines in senile rats

To investigate the regulating effect of Ginkgo biloba extract 50 (GBE50) on pre-inflammatory factors interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and anti-inflammatory factors interleukin-4 (IL-4), interleukin-10 (IL-10) of hippocampus in senile rats, in order to explore the protective mechanism of GBE50 on central nervous system of senile animals. SD rats were randomly divided into four groups: the normal group, the model group, the GBE50 group and the EGB761 group. Rats were intraperitoneally injected with 100 mg x kg(-1) D-galactose every day for 42 days to establish the senile rat model. At the 21st day, the GBE50 group and the EGB761 group were orally administered with 60 mg x kg(-1) for 21 days. IL-1beta mRNA and TNF-alpha mRNA expressions were detected by real-time fluorescence quantitative PCR assay, IL-1beta and TNF-alpha protein expressions were detected by immunohistochemistry, IL-4 and IL-10 protein contents were detected by ELISA. D-galactose caused imbalance between pre-inflammatory factors and anti-inflammatory factors of hippocampus in senile rats, GBE50 and EGB761 reduced IL-1beta mRNA expression (P < 0.05) and TNF-alpha and IL-1beta protein level (P < 0.01) and up-regulated IL-10 protein content (P < 0.01, P < 0.05). The mechanism of GBE50 in protecting central nervous system is probably related to its effect in mitigating inflammatory of central nervous system.

Flagellin Administration Protects Respiratory Tract from Burkholderia cepacia Infection

Burkholderia cepacia is an important pathogen that often causes pneumonia in immunocompromised individuals. Here, it was demonstrated that the TLR5 agonist flagellin could locally activate innate immunity. This was characterized by rapid expressions of IL-1beta, TNF-alpha, and iNOS mRNA and a delay in the expression of IL-10 mRNA. A significant elevation in the IL-1beta, TNF-alpha, and nitric oxide levels was also noted. In the respiratory tract, flagellin induced neutrophil infiltration into the airways, which was observed by histopathological examination and confirmed by the neutrophil count and level of myeloperoxidase activity. This was concomitant with a high activity of alveolar macrophages that engulfed and killed B. cepacia in vitro. The flagellin mucosal treatment improved the B. cepacia clearance in the mouse lung. Thus, the present findings illustrate the profound stimulatory effect of flagellin on the lung mucosal innate immunity, a response that needs to be exploited therapeutically to prevent the development of respiratory tract infection by B. cepacia.

Pre-administration of L-tryptophan improved ADR-induced early renal failure in mice

AimsThe aim of this study was to determine the localization of the rate-limiting enzyme indoleamine 2, 3-dioxygenase (IDO) and its metabolite in mice kidneys after adriamycin (ADR)-induced renal failure. We also examined the effect of l-tryptophan (Trp) administration on this model. Main methodsBALB/c mice were treated with 15mg/kg ADR to induce renal failure. The change of IDO and l-kynurenine (Kyn) following ADR-induced renal failure was examined by immunostaining combined with hematoxylin and eosin (HE) and Periodic acid-Schiff (PAS) staining for morphological analysis, and the concentration of l-Kyn was measured by HPLC. The effect of l-Trp administration on ADR-induced renal failure was also investigated. Key findingsTime-dependent increase of IDO immunostaining was observed in tubular cells from Day 4 after ADR injection. It was found that mice fed with l-Trp had less chance of renal failure at four days after ADR injection than mice untreated with l-Trp, but not at seven days. Further, l-Trp treatment significantly suppressed an increased level of TNF-alpha mRNA, but not of TGF-beta and IL1-beta after renal injury. SignificanceLocal IDO expression in tubules was induced markedly after ADR- induced renal failure. l-Trp administration can be effective in suppressing ADR-induced renal failure at an early stage.

The anti-inflammatory effects of the 5-HT3 receptor antagonist tropisetron are mediated by the inhibition of p38 MAPK activation in primary human monocytes

BackgroundThere is evidence from human and animal research that 5-hydroxytryptamine (5-HT) 3 receptor antagonists, particularly tropisetron, exert analgesic and anti-inflammatory activity. We have demonstrated that tropisetron inhibited lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF)alpha and interleukin-(IL-)1beta release in primary human monocytes. The underlying mechanisms of these effects have not been investigated in detail so far. MethodsThe molecular mechanisms of the anti-inflammatory effects of tropisetron were investigated in human primary monocytes in vitro by studying IL-1beta and TNFalpha mRNA levels by PCR and reporter gene assay and by elucidating the phosphorylation of p38 mitogen activated kinase (MAPK) by Western blot. ResultsThe steady state levels of IL-1beta and TNFalpha mRNA in LPS-activated human peripheral monocytes and the transcriptional activity of the TNFalpha promoter were not inhibited by tropisetron, suggesting that the inhibitory activity of this 5-HT3 receptor antagonist takes place at the post-transcriptional level. Additionally, we found that tropisetron prevents the phosphorylation and thus activation of the p38 MAPK, which is involved in post-transcriptional regulation of various cytokines. ConclusionOur data indicate that the anti-inflammatory effects of the 5-HT3 receptor antagonist tropisetron, as shown in vivo, are possibly mediated by a selective inhibition of pro-inflammatory cytokines at the post-transcriptional level. 5-HT3 receptor antagonists are therefore a new and promising therapeutic option. New and more selective – in respect to the 5-HT3 subtypes – 5-HT3R antagonists might be a future perspective in the pharmacological treatment of inflammatory diseases.

Inhibition of NF-κB-Dependent Cytokine and Inducible Nitric Oxide Synthesis by the Macrocyclic Ellagitannin Oenothein B in TLR-Stimulated RAW 264.7 Macrophages

Immunomodulatory effects of oenothein B (1), a macrocyclic ellagitannin from various Onagraceae species, have been described previously. However, the mechanisms underlying the anti-inflammatory activity of 1 have not been fully clarified. The effects of 1 were investigated on inducible nitric oxide synthase, TLR-dependent and TLR-independent signal transduction cascades, and cytokine expression using murine macrophages (RAW 264.7). Compound 1 (10-60 μg/mL) reduced NO production, iNOS mRNA, and iNOS protein levels in a dose-dependent manner, without inhibition of iNOS enzymatic activity. It reduced the binding of the NF-κB p50 subunit to the biotinylated-consensus sequence and decreased nuclear p65 translocation. Gallic acid as a subunit of the macrocyclic ellagitannin 1 showed a far lower inhibitory activity. Nitric oxide production was reduced by 1 after stimulation using TLR2 (Pam2CSK4) and TLR4 (Kdo2) agonists, but this compound did not inhibit inducible nitric oxide synthesis after stimulation using interferon-gamma. IL-1beta, IL-6, and TNF-alpha mRNA synthesis was clearly reduced by the addition of 1. Oenothein B (1) inhibits iNOS after stimulation with LPS, TLR2, and TLR4 agonists via inhibition of TLR/NF-κB-dependent inducible nitric oxide and cytokine synthesis independent from IFN-gamma/JAK/STAT pathways. The full molecular structure of this macrocyclic ellagitannin seems to be required for its immunomodulatory actions.

Abstract 343: Induction of Apoptosis in Human Macrophages by 7-ketocholesterol and Iron Ascorbate

Inflammation is the new paradigm for the development and progression of atherosclerotic lesions in coronary artery disease. Factors that increase oxidative stress and apoptosis of cells within the lesion contribute to sustaining the inflammatory response. It was hypothesized that redox-active iron and oxysterols, which have both been found in association with atherosclerotic plaques, contribute to oxidative stress and apoptosis by distinct, but overlapping signaling pathways. The human acute monocytic leukemia cell line THP-1 was used as a model system to study the roles of redox-active iron and 7-ketocholesterol in macrophage apoptosis. The ability of both reagents to induce apoptosis in THP-1 macrophages was demonstrated using flow cytometry. The kinetics of poly(ADP-ribose) polymerase (PARP) cleavage as measured by ELISA were also similar. qRT-PCR was used to measure mRNA levels of several pro- and anti-apoptotic genes. While 7-ketocholesterol increased the level of CHOP mRNA (a transcription factor in endoplasmic reticulum (ER)-initiated apoptosis), there was no similar increase in response to iron ascorbate treatment. Expression of other markers of ER stress, like the transcription factors ATF3 and CEBPbeta, was increased by both compounds, but the timing of changes in gene expression was different. TNFalpha regulates both apoptosis and inflammatory cytokine production. Macrophages are major producers of TNFalpha as well as being highly responsive to the cytokine. A comparison of the changes in TNFalpha mRNA and protein levels in response to iron ascorbate and 7-ketocholesterol suggested that while ERK1/2 and NFkB signaling are important in TNFalpha expression, differentially regulated post-transcriptional processes determine the release of TNFalpha by macrophages. These preliminary results suggest that the apoptotic pathways activated by oxysterols and redox-active iron may be different and that these compounds may have additive or synergistic effects on lesion progression.

Expression of TNF-alpha mRNA, but not of TNF-alpha receptors mRNA, is detected in single murine oocyte and decreases during oocyte meiotic maturation: single-cell RT-PCR data

The present study was conducted to investigate the expression of TNF-alpha and its receptors (types I and II) in both oocytes with germinal vesicle and the first polar body in mice. Oocytes with intact germinal vesicle were isolated from mouse ovaries and subjected to in vitro maturation to obtain oocytes forming the first polar body. A reverse transcription polymerase chain reaction (RT-PCR) was used to examine the expression of TNF-alpha and its receptors at mRNA level. mRNA TNF-alpha was expressed in single oocytes and its level was decreasing during transition from germinal vesicle to the first polar body stage. At the same time the expression of TNF-receptors was not observed in single oocyte. These data are the important link in understanding of the molecular mechanisms regulating oocyte maturation as well as follicle development.

Tu1174 Attenuated Progression of Gastric Dysplasia by Administration of Green Tea Catechins in INS-GAS Mice

Backgrounds: Green tea catechins (GTCs) have been shown to provide anti-carcinogenic effects in several laboratory studies. Although the effect of green tea consumption on the risk of gastric cancer has been studied in epidemiological studies, the results remain controversial. The anti-carcinogenic mechanisms of GTCs have not been fully evaluated in rodent models of gastric cancer. Previous reports indicate that transgenic INS-GASmice are hypergastrinemic and induced corpus gastritis and eventually developed gastric cancer at over 20 months old. Aim: The aim of our study was to investigate the molecular effects of GTCs on gastritis and premalignant lesions in INS-GAS mice. Methods: Thirty eight, male INSGAS mice on a FVB background were used in this study. Mice were divided into two groups: A GTCs group and control. Mice in the GTCs group were allowed free access to drinking water containing 2000ppm of GTCs from the age of 9 weeks. Mice were euthanized at the age of 13 or 37 weeks. Histological scores (inflammation and dysplasia) were graded on a blinded basis by pathologist on a scale from 0 to 4. The mRNA expressions of IFN-gamma and TNF-alpha in the corpus of stomachs were evaluated using quantitative PCR. Statistical analysis was performed using a Mann-Whitney U test. Results: Body weights in mice supplemented with GTCs were significantly lower than that of controls at both 13 and 37 weeks of age (p < 0.05). Treated and control mice developed minimal to mild corpus gastritis, although dysplasia was evident. The histological score of dysplasia in mice supplemented with GTCs was significantly lower than that in controls at 37 weeks of age (p < 0.05). GTCs supplementation did not affect the inflammation score. The mRNA level of IFN-gamma in mice administrated GTCs was significantly lower than that of controls at 13 weeks of age (p < 0.05), although it did not reach statistical significance at 34 weeks of age (p = 0.056). GTCs supplementation did not alter TNF-alpha mRNA expression levels between both groups. Conclusions: Administration of GTCs attenuated progression of gastric dysplasia in INS-GAS male mice. These results demonstrated that IFN-gamma dependent mechanisms may be involved in the protective role of GTCs in development of gastritis and premalignant lesions in this rodent model.

Tu1175 The Protective Influence of Vitamin D in Hepatocellular Carcinoma

Backgrounds: Green tea catechins (GTCs) have been shown to provide anti-carcinogenic effects in several laboratory studies. Although the effect of green tea consumption on the risk of gastric cancer has been studied in epidemiological studies, the results remain controversial. The anti-carcinogenic mechanisms of GTCs have not been fully evaluated in rodent models of gastric cancer. Previous reports indicate that transgenic INS-GASmice are hypergastrinemic and induced corpus gastritis and eventually developed gastric cancer at over 20 months old. Aim: The aim of our study was to investigate the molecular effects of GTCs on gastritis and premalignant lesions in INS-GAS mice. Methods: Thirty eight, male INSGAS mice on a FVB background were used in this study. Mice were divided into two groups: A GTCs group and control. Mice in the GTCs group were allowed free access to drinking water containing 2000ppm of GTCs from the age of 9 weeks. Mice were euthanized at the age of 13 or 37 weeks. Histological scores (inflammation and dysplasia) were graded on a blinded basis by pathologist on a scale from 0 to 4. The mRNA expressions of IFN-gamma and TNF-alpha in the corpus of stomachs were evaluated using quantitative PCR. Statistical analysis was performed using a Mann-Whitney U test. Results: Body weights in mice supplemented with GTCs were significantly lower than that of controls at both 13 and 37 weeks of age (p < 0.05). Treated and control mice developed minimal to mild corpus gastritis, although dysplasia was evident. The histological score of dysplasia in mice supplemented with GTCs was significantly lower than that in controls at 37 weeks of age (p < 0.05). GTCs supplementation did not affect the inflammation score. The mRNA level of IFN-gamma in mice administrated GTCs was significantly lower than that of controls at 13 weeks of age (p < 0.05), although it did not reach statistical significance at 34 weeks of age (p = 0.056). GTCs supplementation did not alter TNF-alpha mRNA expression levels between both groups. Conclusions: Administration of GTCs attenuated progression of gastric dysplasia in INS-GAS male mice. These results demonstrated that IFN-gamma dependent mechanisms may be involved in the protective role of GTCs in development of gastritis and premalignant lesions in this rodent model.

Abstract 5659: In vivo efficiency and biodistribution of TNF-alpha expressing small-sized vector DNA after intratumoral nonviral jet-injection

Abstract Nonviral gene therapy is gaining growing importance and has been applied in numerous studies to treat cancer. Plasmid vectors are still used in the majority of cancer gene therapy approaches. However, it is broadly accepted, that plasmid-vectors need significant improvements to enhance their safety and efficiencies. In this context small-sized vectors are attractive alternatives. The MIDGE-platform (Mologen, Berlin, Germany) represents such small vector system harboring the essential expression cassette in a double-stranded linear DNA molecule and end-sealing oligonucleotide loops. We used the MIDGE-vector for the expression of the human TNF-alpha. Here we show, that this MIDGE-CMVhTNF system permits significantly improved in vivo TNF-alpha expression in the A375 and MeWo human melanoma xenotransplant model after intratumoral in vivo jet-injection gene transfer in nude mice. The comparison to the respective plasmid-vector revealed an up to 22-fold increase of the MIDGE-CMVhTNF mediated intratumoral TNF-alpha expression. Apart from these efficiency studies the safe use of such minimal-size vector is essential. We therefore analyzed the biodistribution and clearance of the MIDGE-CMVhTNF vector after jet-injection in A375 melanoma bearing nude mice. For this, the animals received 5 jet-injections through the skin into the tumor at a pressure of 2.8 Bar. Each jet-injection delivered 0.01 mL MIDGE-DNA at a concentration of 1mg/mL. The intratumoral MIDGE-DNA load was analyzed 5, 10, 20, 40 minutes and 3, 6, 24, 48, 72 hours as well as 7 days after jet-injection by quantitative real-time PCR. For the same time points the systemic dissemination of the MIDGE-DNA was quantitatively analyzed by real-time PCR in the blood, liver, lung, spleen, kidney, ovary, heart and brain. The analyses show high DNA-load of up to 12 ng vector-DNA / 250 ng tumor DNA and a clearance of this DNA in the tumors within 3 to 7 days. This is paralleled with the decline of TNF-alpha expression. Regarding systemic biodistribution MIDGE-DNA levels in blood samples are peaking at 30 minutes after jet-injection, followed by the rapid drop within 60 minutes after DNA application. The MDGE-vector DNA was also detected in liver, lung, spleen, kidney, brain, ovary and heart at varying pg-levels for up to 3 hours after jet-injection. This was followed by the clearance of this DNA within 6 to 24 hours after gene transfer. More importantly, in none of these organs TNF-alpha mRNA expression was detected by real-time RT-PCR at any time point. In summary these data indicate, that the MIDGE-CMVhTNF vector leads to improved in vivo transgene expression and shows only limited systemic biodistribution after intratumoral jet-injection, associated with its rapid clearance. These parameters are of importance for the potential clinical use of this vector. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5659. doi:1538-7445.AM2012-5659