TNF-alpha mRNA Expression Research Articles

Therapeutic potential of seaweed extracts: In vitro and in vivo studies on alleviating inflammation and enhancing intestinal barrier function.

Rich in diverse compounds such as polysaccharides, pigments, and phenols, seaweed offers not only nutritional benefits but also functional elements such as fucoidan, which demonstrates anticoagulant, anticancer, and anti-inflammatory properties. This investigation screened 22 seaweed extracts obtained using either water or ethanol as solvents from a total of fourteen seaweed species to evaluate their potential as dietary supplement ingredients. In inflammation-induced Caco-2 cell models, Saccharina japonica, Capsosiphon fulvescens, Hizikia fusiforme, and Chondrus ocellatus exhibited a significant increase in TEER values 24 h after treatment with seaweed extracts, in contrast to the cocktail group (p < 0.05). Additionally, permeability significantly decreased compared to the cocktail group (p < 0.05). Notably, these species exhibited a reduction in mRNA expression of TNF-alpha and iNOS, coupled with an elevation in the protein expression of tight junction proteins. Moreover, when tested in DSS-induced mice, these seaweeds demonstrated enhancements in colon health indices, suggesting their potential as beneficial treatments for inflammatory bowel diseases and associated conditions. This study demonstrates the therapeutic promise of these four seaweed species and emphasizes their prospective role in treating inflammatory bowel diseases, urging further exploration of their therapeutic mechanisms and clinical applications.

Molecular Approach to Identify Anti-inflammatory Potential of Stevioside in HFD-induced Type 2 Diabetic Rats: Evidence From in Vivo Study

Stevioside is a natural sweetener derived from the leaves of the Stevia rebaudiana plant. It has gained popularity as a sugar substitute due to its intense sweetness without adding calories or affecting blood sugar levels, making it a suitable option for people with diabetes or those looking to reduce their sugar intake. Studies have shown that stevioside has glucose lowering effects. Previous studies have shown that it has significant role in skeletal muscle but its role on expression of inflammatory signaling molecules in adipose tissue against high diet and sucrose-induced type-2 diabetes in experimental rats is yet to be done. The current research was undertaken to investigate if stevioside could also exert its antidiabetic effects by circumventing adipocyte induced inflammation, a key driving factor for insulin resistance in obese individuals. Effective dose of stevioside (20 mg/kg b.wt) was administered orally for 45 days to high fat diet and sucrose induced type-2 diabetic rats. Interestingly, stevioside treatment restores the elevated serum levels of proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and sterol regulatory element binding protein-1c (SREBP-1c) and enhances Peroxisome Proliferator–activated receptor-γ (PPAR-γ) in adipocytes of diabetic rats. The gene expression of IR, GLUT4 and PPAR-γ mRNA were also significantly activated in stevioside treated groups but reduced IL-1 beta, IlL-6, IKKB, TNF-alpha and NFkB mRNA expression in diabetic adipose tissue. More importantly, stevioside acts very effectively as metformin to circumvent inflammation and insulin resistance in diabetic rats. Our results clearly show that stevioside inhibits obesity induced insulin resistance by ameliorating the inflammatory events and upregulating insulin signalling molecules. Keywords: Stevioside, HFD-T2DM, pro inflammation, insulin signalling; adipose tissue, obesity; signaling pathways; Therapautics.

Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles Modulate Apoptosis, TNF Alpha and Interferon Gamma Response Gene mRNA Expression in T Lymphocytes.

Recent studies have highlighted the therapeutic potential of small extracellular bodies derived from mesenchymal stem cells (MSC-sEVs) for various diseases, notably through their ability to alter T-cell differentiation and function. The current study aimed to explore immunomodulatory pathway alterations within T cells through mRNA sequencing of activated T cells cocultured with bone marrow-derived MSC-sEVs. mRNA profiling of activated human T cells cocultured with MSC-sEVs or vehicle control was performed using the QIAGEN Illumina sequencing platform. Pathway networks and biological functions of the differentially expressed genes were analyzed using Ingenuity pathway analysis (IPA)® software, KEGG pathway, GSEA and STRING database. A total of 364 differentially expressed genes were identified in sEV-treated T cells. Canonical pathway analysis highlighted the RhoA signaling pathway. Cellular development, movement, growth and proliferation, cell-to-cell interaction and inflammatory response-related gene expression were altered. KEGG enrichment pathway analysis underscored the apoptosis pathway. GSEA identified enrichment in downregulated genes associated with TNF alpha and interferon gamma response, and upregulated genes related to apoptosis and migration of lymphocytes and T-cell differentiation gene sets. Our findings provide valuable insights into the mechanisms by which MSC-sEVs implement immunomodulatory effects on activated T cells. These findings may contribute to the development of MSC-sEV-based therapies.

Treatment of Ischemic Stroke by Atorvastatin-Loaded PEGylated Liposome.

There is insufficient evidence on the effect of nanoparticles, particularly liposomes loaded with a statin, on acute ischemic stroke. We investigated the impact of atorvastatin-loaded PEG (polyethylene glycol) conjugated liposomes (LipoStatin) on the outcomes in rats with cerebral ischemia-reperfusion. PEGylated liposome loaded with atorvastatin was developed as a nanoparticle to specifically accumulate in an ischemic region and release the drug to ameliorate the harmful effects of the stroke. LipoStatin was administered to rats with transient middle cerebral artery occlusion through the tail vein immediately after reperfusion (LipoStatin group). LipoStatin efficiently accumulated at the cerebral ischemic injury site of the rat. The LipoStatin group showed a significantly reduced infarct volume (p < 0.01) in brain micro-MR imaging and improved neurological function recovery compared to the control group (p < 0.05). In addition, markedly improved brain metabolism using fluorine-18 fluorodeoxyglucose micro-PET/CT imaging was demonstrated in the LipoStatin group compared with the control group (p < 0.01). Mechanistically, as a result of evaluation through IL-1 beta, TNF-alpha, ICAM-1, and Iba-1 mRNA expression levels at 5days after cerebral ischemia, LipoStatin showed significant anti-inflammatory effects. Protein expression of occludin, JAM-A, Caveolin-1, and eNOS by western blot at 3days and fluorescent images at 7days showed considerable recovery of blood-brain barrier breakdown and endothelial dysfunction. PEGylated LipoStatin can be more effectively delivered to the ischemic brain and may have significant neuroprotective effects. Thus, PEGylated LipoStatin can be further developed as a promising targeted therapy for ischemic stroke and other major vascular diseases.

Effects of systemic androgens on late-stage urethral wound healing: An animal study.

There are limited data regarding the effects of systemic androgens on late-stage urethral wound healing. To evaluate the effects of systemic androgens on fibrosis and scar formation in late-stage urethral wound healing. Forty-five male Sprague Dawley rats were divided into three groups. First group consisted of 15 rats that were castrated on 23days of age and were given 5mg/kg testosterone undecanoate with 1/25ml cottonseed oil intraperitoneally at weekly intervals for 3weeks (castrated and replaced with testosterone rats [CAS+T] group). The castrated rats (CAS) group included 15 castrated rats. The remaining 15 rats underwent sham surgery. CAS and sham groups also received 1/25ml cottonseed oil intraperitoneally at weekly intervals for 3weeks. Furthermore, all groups were divided into three subgroups after testosterone/placebo administration (urethroplasty performed after first, second, and third weeks) in accordance with the urethroplasty timing. All animals were sacrificed 6weeks after urethroplasty. Serum testosterone level was measured, tissue samples were investigated using hematoxylin and eosin and Masson's trichrome. Alpha-SMA, Coll 1 and Coll 3 primary antibodies were applied for immunohistochemical examination. Expression of cytokines and growth factors, such as Bax, Bcl2, IL-10, IP-10, TNF-alpha, TGFb1, MMP9, Col-I, Col-III, TIMP-1, fibronectin, fibroblast growth factor 10, platelet-derived growth factor, alpha-SMA, were also evaluated in the tissues. The blood testosterone levels were significantly higher in CAS+T group at the time of urethroplasty compared with the levels in CAS group; however, this difference was not observed at the time of sacrification (p<0.001 and 0.97, respectively). Histological analysis with hematoxylin and eosin and Masson's trichrome staining revealed a significantly higher fibrosis in the sham group compared with the others. Significantly lower fibrosis was detected in the CAS group in the pairwise comparison of the pathological fibrosis area between the CAS and CAS+T groups (p<0.001). Furthermore, tissue collagen-1, collagen-3, and alpha-SMA expression levels were statistically different between CAS and CAS+T groups (p<0.001, <0.05, and <0.001, respectively). The tissue levels of BAX, TIM-1, MMP-9, Coll-I, Coll-III, TGF-beta, TNF-alpha, and IL-10 mRNA expressions in the CAS+T group were different than the levels in CAS group (as <0.5-fold and >1.5-fold changes, respectively). The expressions of all these markers were significantly higher in the sham group. The subgroup analysis of CAS+T group (urethroplasty performed after first, second, and third weeks) revealed similar histopathological wound healing findings. Debate continues on the effects and benefits of androgen use regarding urethral healing. There are two main routes for administration as systemic or local. This study focuses on the late-stage histologic and biochemical effects of systemic androgens. Systemic androgens adversely affect wound healing and cause abnormal extracellular matrix as well as scar formation.

Efficacy of Nitric Oxide-Releasing Nanofibers in Reducing Renal Ischemia-Reperfusion Injury in a Rat Model.

BackgroundThis study aimed to analyze the preventive effect of nitric oxide (NO)-releasing nanofibers against ischemia-reperfusion injury (IRI) and to determine the mechanism of action as a novel NO delivery system in a rat model.Material/MethodsEight-week-old male Sprague-Dawley rats, weighing 250 to 280 g, were divided into 3 groups: sham, untreated (n=5); control, renal ischemia injury for 55 min (n=4); and NO24, renal ischemia injury for 55 min with kidney wrapping of NO-releasing nanofiber for 24 h (n=6). mRNA expression was measured by real-time polymerase chain reaction (PCR), whereas protein expression was assessed by immunohistochemistry and western blot analysis.ResultsSerum creatinine levels in the sham, control, and NO24 groups were 0.48±0.08, 4.66±0.33, and 2.60±1.00 mg/dL, respectively (P=0.002). Anti-apoptotic Bcl-2 protein expression differed significantly between the control and the NO24 groups (Bcl-2/β-actin; control, 0.50±0.12; NO24, 1.56±0.56; P=0.024). mRNA expression level of the inflammatory cytokine tumor necrosis factor-α (TNF-α) was significantly higher in the control group (23.24±11.32, P=0.016) than in the sham group (1.00±1.21), and mRNA expression of TNF-α in the NO24 group (1.28±1.16, P=0.010) was significantly lower than in the control group. Histological analysis revealed decreased atrophy and necrosis in the NO24 group compared to those in the control group.ConclusionsThis study demonstrated that kidney wrapping of NO-releasing nanofibers had a protective effect against kidney IRI through anti-apoptotic and anti-inflammatory mechanisms.

MO690HIGHER EXPRESSION OF TOLL LIKE RECEPTOR AND CELL ADHESION MOLECULES ARE ASSOCIATED WITH RECURRENT/RELAPSE PERITONITIS AND MORTALITY IN PATIENT ON CAPD

Abstract Background and Aims CAPD is an established modality of treatment for patients with end stage renal disease (ESRD). Peritonitis is a leading cause of technique failure and death in patients on CAPD. Studies on expressions of host genetic factors like TLRs, CAMs, Pro and Anti-inflammatory cytokines and their link to peritonitis and other co-morbidity and functional status are lacking throughout the world. Hence the present study was done to determine the expressions of TLR2 and TLR4, CAMs and inflammatory cytokines in patients on CAPD. Method A total of 110 ESRD patients categorized into 3 groups: 1- CAPD patients (n=45), group 2- CAPD with peritonitis patients (n=35), and group 3- CAPD patients with recurrent/relapsing peritonitis. (n=30) from department of Nephrology, Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Raebareli Road, Lucknow were included in the study. Blood sample was collected for analysis of cytokines and Cell adhesion molecules level and RNA extraction. Dialysate (only from patients undergoing PD with or without peritonitis)- whole drained bag; 50-100 ml for microscopy and culture for bacterial and fungal pathogens using automated BACTEC culture system in patients with peritonitis and supernatant was collected for cytokine ELISA. Statistical analysis: One-way ANOVA (Two tailed) and non-parametric student t-test used for statistical analysis of differences between the group. Results mRNA expression of TLR 2 and TLR 4 expression was higher in peritonitis and recurrent/relapsing peritonitis group as compared to CAPD patient without peritonitis group. mRNA expression of TNF-alpha, IL-1B and IFN-gamma was higher in peritonitis group of patients compared to all other group of patients. mRNA expression of IL-10 and IL-4 was also highest in Peritonitis group. Level of IL-1β and TNF-alpha was observed to be elevated in peritonitis and recurrent/relapse peritonitis patients compared to CAPD in sera and dialysate. IL-12 level in Sera was also elevated in peritonitis patients but its level in dialysate was highest in CAPD without peritonitis as compared to peritonitis and recurrent/relapse. IL-10 and IL-4 was observed to be elevated in peritonitis group followed by CAPD with recurrent/relapse peritonitis in sera whereas in dialysate level of IL-4 was higher in both peritonitis group and Recurrent groups of patients but IL-10 level was highest in peritonitis group but less in recurrent/relapse. mRNA Expression levels of adhesion molecule ICAM-1 and LFA-1 were observed to be higher in Peritonitis and Recurrent groups as compared to the CAPD without peritonitis; whereas, in sera the level of ICAM-1 was higher in peritonitis patients. However, in dialysate its level was observed to be almost similar. Level of LFA-1 was observed to be higher in peritonitis group in dialysate but in sera its level was higher in Recurrent group with peritonitis. Conclusion It is likely that CAMs are also involved in the pathogenesis of CAPD peritonitis, its morbidity and mortality. TLRs activation by bacterial molecules leads to the induction of chemokines and cytokines through the activation of NF-ķB pathway and may be responsible for atherosclerosis, morbidity, and mortality in ESRD patients. Elevated level of ICAM-1, IL-1beta, TNF-a and IFN-G may be responsible for chronic inflammation in PD patients.

Knockout of the Cannabinoid Receptor 2 Gene Promotes Inflammation and Hepatic Stellate Cell Activation by Promoting A20/Nuclear Factor-κB (NF-κB) Expression in Mice with Carbon Tetrachloride-Induced Liver Fibrosis.

BackgroundThis study aimed to investigate the effect of deleting the cannabinoid receptor 2 (CB2) gene on the development of hepatic fibrosis induced by carbon tetrachloride (CCl4) in mice via regulating inflammation.Material/MethodsThe DNA was extracted from the tails of mice to identify whether the cannabinoid receptor 2 gene was successfully knocked out. A liver fibrosis model was established by an intraperitoneal injection of CCl4 into mice. Hepatic damage and hepatic fibrosis were evaluated by detecting serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and staining paraffin sections of liver tissue with hematoxylin-eosin (HE). The secretion and distribution of collagen in liver tissue were observed by Masson staining. Western blot analysis was performed to detect the expression of α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), tumor necrosis factor alpha-induced protein 3 (A20), phosphorylated nuclear factor-κB p65 (p-NF-κB p65), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6) in liver tissue. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of IL-6 and TNF-α mRNA in liver tissue.ResultsCompared with the control mice, the mice with CB2 knockout that were exposed to CCl4 exhibited increased liver damage, liver fibrosis, and upregulated α-SMA, TGF-β1, A20, and p-NF-κB p65 protein levels. IL-6 and TNF-α protein levels and mRNA levels were upregulated.ConclusionsThe deletion of the CB2 gene promoted the activation of hepatic stellate cells in mice with liver fibrosis and aggravated liver fibrosis by up-regulating the protein expression of A20 and p-NF-κB p65 and inducing inflammatory response, potentially providing new insight into the treatment of liver fibrosis.

Abstract 547: Hur Dependent Regulation of Inflammatory Remodeling Following Myocardial Ischemia/reperfusion Injury

Myocardial infarction (MI), resulting from ischemia/reperfusion (I/R) injury due to the obstruction of coronary blood flow, affects an estimated 800,000 Americans annually resulting in a cost of nearly 12 billion dollars to the health care system. Recent medical advances have led to improved survival following acute MI, but unfortunately, the post-MI inflammatory and fibrotic remodeling of the heart is driving an increase in the prevalence of heart failure. Preliminary data from our lab shows that the RNA binding protein Human antigen R (HuR) undergoes nuclear-to-cytoplasmic translocation within 30 minutes and is increased in expression at both 2 hours and 7 days post-I/R. The objective of this work is to determine the mechanistic role of HuR on the regulation of cardiac cytokine/chemokine expression and the functional effect of HuR on macrophage infiltration and polarization post-I/R. Wild-type mice were subjected to 30 minutes of LAD (left anterior descending) coronary artery ligation (ischemia) followed by reperfusion with HuR inhibitor or vehicle control administered at the start of reperfusion. Our results show that inhibition of HuR reduces cardiac mRNA expression of IL-6, TNF-alpha, and ICAM-1 at 2 hours post-reperfusion, but does not affect initial (24 hour) infarct size. In addition, HuR inhibition reduces macrophage infiltration at 7 days post-I/R, as well as cardiac fibrosis and ventricular dilation while preserving cardiac function at 14 days post-I/R. Furthermore, H9c2s treated with HuR inhibitor blunts the mRNA expression of CCL2 after acute stimulation of lipopolysaccharide. In conclusion, we show that HuR inhibition at the time of reperfusion is protective against post-MI inflammatory and fibrotic remodeling through a reduction in inflammatory gene expression and macrophage infiltration. Previous work by the lab has demonstrated a direct role for HuR in cardiac myocytes, but these results also suggest that HuR-mediated gene expression in macrophages may play a role in post-MI inflammatory and fibrotic remodeling.

Epigenetic regulation of TLR2‐mediated periapical inflammation

To determinethe methylation pattern of TLR2 gene promoter and its association with the transcriptional regulation of periapical inflammatory and angiogenic responses in symptomatic and asymptomatic forms of apical periodontitis. In this cross-sectional study, apical lesions were obtained from volunteers with asymptomatic apical periodontitis (AAP) (n=17) and symptomatic apical periodontitis (SAP) (n=17) scheduled for tooth extraction, and both total RNA and DNA were extracted. DNA was bisulfite-treated, a region of CpG island within the TLR2 gene was amplified by qPCR and the products were sequenced. Additionally, the mRNA expression of TLR2, TLR4, IL-6, IL-12, TNFalpha, IL-23, IL-10, TGFbeta, VEGFA and CDH5 was analysed by qPCR. The data were analysed with chi-square tests, Mann-Whitney or unpaired t-tests, and Spearman´s correlation; variable adjustments were performed using multiple linear regression (P<0.05). TLR2 depicted a hypomethylated DNA profile at the CpG island in SAPwhen compared with AAP, along with upregulated expression of TLR2, with pro-inflammatory cytokines IL-6 and IL-23, and the angiogenesis marker CDH5 (P<0.05). TLR2 methylation percentage negatively correlated with mRNA levels of IL-23 and CDH5 in apical periodontitis. Lower methylation frequencies of single CpG dinucleotides -8 and -10 localized in close proximity to nuclear factor κB (NFκB) binding within the TLR2 promoter were identified in SAP versus AAP (P<0.05). Finally, unmethylated -10 and -8 single sites demonstrated up-regulation of IL-23, IL-10 and CDH5 transcripts compared to their methylated counterparts (P<0.05). TLR2 gene promoter hypomethylation was linked to transcriptional activity of pro-inflammatory cytokines and angiogenic markers in exacerbated periapical inflammation. Moreover, unmethylated single sites in close proximity to NFκB binding were involved in active transcription of IL-23, IL-10 and CDH5.

P0123THE IMPORTANCE OF PPAR-ALPHA IN AKI TO CKD TRANSITION IN NEPHROPATHY INDUCED BY FOLIC ACID

Abstract Background and Aims PPAR-alpha is a nuclear receptor which plays major role in the regulation of lipid metabolism, activation of PPAR-alpha has been shown to have beneficial effects in renal diseases. Acute kidney injury (AKI), even if followed by renal recovery, is a risk factor for the future development of chronic kidney disease (CKD) and end-stage renal disease (ESRD). Our main objective was to determine the importance of PPAR-alpha in AKI to CKD transition in folic acid induced nephropathy. Method Male C57/Bl6 and PPARKO (C57Bl6 background) mice were divided in 4 groups, Control, Folic Acid, Gemfibrozil + Folic Acid and PPARKO + Folic Acid. Animals has been treated with single dose of Folic Acid (250mg/kg i.p) and Gemfibrozil (150mg/kg gavage) euthanized after 48 hours for AKI assessment and 28 days for CKD. Renal parameters, histology, real time PCR were performed to investigate renal injury, inflammation and fibrosis. Results After 48 hours of folic acid inducing AKI, PPARKO mice showed decreased urea levels (Folic Acid: 178,9±25,2 PPARKO: 92,4±15,5), less tubular injury (Folic Acid: 0,61±0,05 PPARKO: 0,21±0,03), lower mRNA expression of NGAL (Folic Acid; 32,2±7,67 PPARKO 3,74±1,71), KIM-1 (Folic Acid: 671,8±170,2 PPARKO: 43,4±29,7) and TNF-alpha (Folic Acid: 2,81±0,58 PPARKO: 0,67±0,14), while PPAR-alpha activation with gemfibrozil showed to have no effects in AKI. After 28 days of folic acid inducing CKD. PPARKO showed same levels of injury than folic acid alone treated mice, however PPAR-alpha activation with gemfibrozil decreased urea levels (Folic Acid: 79,2±3,2 Gemfibrozil: 46,1±3,8), showed less tubulointerstitial fibrosis (Folic Acid: 0,60±0,05 Gemfibrozil: 0,18±0,02) and lower urine protein/creatinine ratio (Folic Acid: 5,48±0,58 Gemfibrozil: 2,53±0,12), Conclusion PPAR-alpha deletion protects against AKI induced by folic acid but did not showed protection in chronic phase, in the other hand ppar-alpha activation with gemfibrozil did not protect in AKI but show to be effective in CKD.

Paternal High Fat Diet and Exercise Regulate Sperm miRNA to Alter Placental Inflammation and Nutrient Transporter Expression in a Sex-Dependent Manner

Abstract Objectives We have shown that male offspring (F1) of fathers (F0) fed a high fat (HF) diet and exercised had greater skeletal muscle insulin signaling and reduced T2DM risk compared to fathers fed HF diet and remain sedentary. The current study extends this work by hypothesizing that F0 HF diet and exercise regulate F1 T2DM risk by early alterations in epigenetics of placental tissue growth via changes in sperm miRNA expression. Methods To test these hypotheses, three-week old male C57BL/6 mice were fed a normal-fat (NF) diet (16% fat) or a HF diet (45% fat) and assigned to either voluntary wheel running exercise or cage activity for 3 months prior to mating with NF diet fed dams. F0 sperm and placental tissue samples were collected to determine changes in placental and fetal weights, placental gene expression, and F0 sperm miRNA expression. Results F0 sperm miRNA 193b expression was decreased while miRNA 204 was increased by paternal exercise. Protein expression of di-methylated histone 3 lysine 9 was decreased with F0 HF diet. Placental and fetal tissue weights were decreased by F0 HF diet in F1 males while no changes in the F1 females. Placental proinflammatory cytokine mRNA expression, including IL-1 beta and TNF-alpha, was reduced by paternal exercise while nutrient transporter mRNA expression was decreased by paternal HF diet only in the placentae of F1 females. Treatment of primary placental cell with miRNA 193 inhibited TNF-alpha mRNA expression. In addition, treatment of the same cells with TNF-alpha increased SLC6a19. Moreover, paternal exercise increased body weight at weaning in a female offspring. Conclusions These results demonstrate that placental tissue weight, placental nutrient transporter gene expression and fetal weights are altered by paternal exercise while placental inflammatory gene expression are influenced by paternal exercise in offspring in a sex-specific manner. Funding Sources This work was supported by USDA ARS Project #3062–51,000-054–00D.

Deep Infiltrating Endometriosis and Activation and Memory Surface Markers and Cytokine Expression in Isolated Treg Cells.

It is not yet clear whether regulatory T (Treg) cells are active, and whether they play a favorable or adverse effect on endometrial foci implantation. Our aim was to evaluate activation and memory surface markers in Treg isolated from peritoneal fluid (PF) and peripheral blood (PB) of women with deep endometriosis and to assess its cytokine mRNA expression. This case-control study included 49 women with deep infiltrating endometriosis and 20 healthy controls. It was analyzed PF and PB of both groups. Cell surface markers GITR, TNFRII, HLA-DR, ICOS, CTLA-4, CD45RA, and CD45RO were evaluated in Treg (CD3+CD4+CD25+CD127lowFoxp3+) cells by flow cytometry. Additionally, Foxp3, TGF-beta, IL-10, IL-6, and TNF-alpha mRNA expression was assessed by real-time PCR in Treg cells (CD4+CD25+CD127dim/-) isolated using magnetic microbeads. Women with endometriosis had higher percentages of TNFRII+ Treg and CTLA-4+ Treg in their PB, and lower percentages of ICOS+ Treg and CD45RO+ Treg in their PF. The groups displayed no differences in mRNA expression. Regardless of the group, in PF, the percentage of Treg cells overall and of CD45RA+ Treg cells were significantly lower, whereas the percentage of TNFRII+ Treg and CD45RO+ Treg were significantly higher than in PB. Foxp3 and TGF-beta mRNA expression were also higher in PF than in PB. Our results indicated that Treg cells in women with endometriosis have a distinct profile of activation and memory markers, but similar cytokine expression. Moreover, we could observe clearly that Treg cells have distinct profile regarding their origin site.

P748Afterload-induced TRIF-dependent signaling promotes fibrosis but not cardiomyocyte hypertrophy

Abstract Background Pressure overload dependent cardiac remodeling is characterized by a continuous functional loss of cardiomyocytes, cardiomyocyte hypertrophy, cell death and fibrosis. The relevance of the MyD88- and NFκB-independent inflammatory TRIF (TIR-domain-containing adapter protein inducing IFN-β) signaling pathway in myocardial hypertrophy is incompletely understood. Methods and results The murine model of afterload induced myocardial hypertrophy (trans-aortic constriction, TAC) was used to investigate the time dependent chemokine expression in C57Bl/6J Wild type (WT) mice after intervention. Myd88- as well as TRIF-dependent chemokines reached their expression maximum at d7 post intervention. This time point was determined for further investigations on TRIF knockout-mice (TRIF−/−) compared to WT mice. While left ventricular (LV) mRNA-expression of TNFalpha and IL6 was upregulated in TRIF−/− mice, CXCL10, CXCL11, RANTES and pathway molecule IRF3 remained unchanged. In the stage of established hypertrophy (35 days after TAC) cytokine levels returned to baseline in WT as well as in TRIF−/− mice. Cellular hypertrophy (increase in cardiomyocyte size) as well as echocardiographic determined increase of LV mass was similar in TRIF−/− and WT mice at d7 as well as at d35 after TAC. Additionally at d7 and d35 the fractional shortening function didn't show any differences between the groups after TAC. On the other hand LV interstitial fibrosis (determined by collagen content) was significantly less distinct in TRIF−/− mice (1.4±0.2%) than in WT mice (2.3±0.3%, (p&lt;0.01 TRIF−/− vs. WT) at d7 following TAC. According to that TGFbeta protein expression was more pronounced in WT (318±85% of sham-operated controls) than in TRIF−/− mice (111±15% of sham-operated controls; p&lt;0.05 TRIF−/− vs. WT). These differences in fibrosis and TGFbeta remained at d35. Even the hydroxyproline concentration was higher in LV tissue of the WT mice than in the TRIF−/− mice d35 after TAC (WT: 93.4±7.7μM, TRIF−/− 54.8±9.0μM, p&lt;0.5 TRIF−/− vs. WT). The expression of TGFbeta signaling pathway associated SMAD proteins (SMAD 3, pSMAD3, SMAD4) was up to two fold higher in WT mice than in TRIF−/− mice. Additionally expression of antifibrotic miR29b was more pronounced in TRIF−/− mice (2.34±0.71) than in WT mice (0.98±0.08) after TAC d7. Conclusion In afterload induced hypertrophy TRIF-dependent signaling doesn't influence cardiomyocyte hypertrophy. But TRIF plays an important role in the regulation of fibrosis in pressure overload dependent remodeling. Through the modulation of the TGF-beta signaling pathway and antifibrotic microRNAs TRIF signaling is involved in the development of interstitial fibrosis. Especially in the early stage of the cardiac remodeling. Acknowledgement/Funding KFO196

Abstract 772: MicroRNA regulatory networks: Concomitant in situ detection of microRNA, mRNA and protein in cancer samples

Abstract The canonical mode of action of microRNAs involves the binding to the 3'UTR of mRNAs thereby mediating repression of protein synthesis. MicroRNAs comprise an interesting new group of cancer biomarkers and may also be therapeutic targets in cancer and other diseases using antisense oligos (ASO). The visualization of potential molecular dynamics between a microRNA, its target mRNA and the effect of protein expression can be facilitated by combined detection in tissue sections. MicroRNAs, as well as ASOs, can be detected in tissue sections or cultured cells by in situ hybridization (ISH) using LNA probes. We have developed a novel ISH assay for microRNA detection with improved sensitivity and signal-to-noise that can be used with any LNA probe on both FFPE and frozen sections. The assay is based on double-labeled LNA™ probes and detection can be either fluorescence or chromogenic staining. We have combined the fluorescence assay with immunofluorescence, for double and triple staining. We also combined the microRNA ISH assay with mRNA in situ hybridization using the highly specific RNAscope probes. We show a variety of combinations, including examples of combined staining of microRNA-17, IL-1beta mRNA or TNF-alpha mRNA, and cytokeratin proteins in automated triple staining procedures of colon cancer samples. The focal expression of IL-1beta mRNA or TNF-alpha mRNA and the loss of microRNA-17 at the invasive front involving inflammatory cells and budding cancer cells is discussed. Citation Format: Boye Schnack Nielsen. MicroRNA regulatory networks: Concomitant in situ detection of microRNA, mRNA and protein in cancer samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 772.

Paternal High Fat Diet and Exercise Differentially Regulate Placental Development and Inflammation in a Sex-Specific Manner in C57BL6/J Mice (P19-003-19)

ObjectivesWe have shown that increases in T2D risk in male offspring when the father consumes a high-fat (HF) diet can be normalized when the father also exercises during preconception, and that this protection may occur by epigenetic increases in insulin signaling within offspring skeletal muscle. In our current study, we investigated to determine how paternal HF diet and exercise conditions alter sperm miRNA, fetal weight and placental inflammation. MethodsThree-week old male C57BL/6 mice were fed a normal-fat (NF) diet (16% fat) or a HF diet (45% fat) and assigned to either voluntary wheel running exercise or cage activity for 3 months prior to mating with NF diet fed dams. Sperm samples were collected to determine changes in miRNA that may account for the enhanced offspring skeletal muscle responses that helped normalize paternal HF-induced glucose intolerance. Placentae were collected to determine whether changes in sperm miRNA expression differed by amount of placental inflammation. ResultsSperm expression of miRNA 193b increased with paternal HF and exercise. In F1 males, placental and fetal weight decreased with HF diet while, in F1 female, paternal HF and exercise had no effect on placental and fetal weights. Paternal HF diet decreased placental IL-6 and TNF-alpha mRNA expression in F1 females, while no effects were observed in F1 male placenta. ConclusionsTaken together these data suggest that paternal HF diet has a greater impact on placental development of male fetuses while paternal exercise has greater impact on placental inflammation of female fetuses. For both female and male fetuses, these paternal influences are mediated via sperm miRNA 193b. miR-193b is involved in regulation of the cell cycle and adipogenesis but may have additional functions. Thus, the exact role of sperm miRNA 193b in sex-specific epigenetic transmission of paternal HF diet and exercise on placental and fetal development needs further evaluation. Funding SourcesUSDA Agricultural Research Service Project #3062-51000-052-00D.

Lack of association between 4 key TNF-alpha promoter polymorphisms and hepatitis C virus infection in a population of Egyptian patients

Introduction: With 170 million chronic hepatitis C virus (HCV) cases worldwide, HCV is considered a major life-threatening pathogen. HCV is a crucial causative of liver cirrhosis and hepatocellular carcinoma. Tumor necrosis factor-alpha (TNF-alpha) is thought to be a mediator in the development of viral hepatitis. Because HCV is epidemic in Egypt, this study aimed to characterize the distribution of TNF-alpha gene promoter polymorphisms and their relation to TNF-alpha expression in HCV patients.&#x0D; Methods: Four promoter polymorphisms; −1031T/C, −863C/A, −857C/T, and −308G/A, were studied by restriction fragment length polymorphism in a population of Egyptian HCV patients.&#x0D; Results: Compared to healthy subjects, none of these polymorphisms were associated with HCV infection. The wild-type −1031T, −863A, −857C, and −308G alleles were highly prevalent in the studied population. Sequencing the promoter region spanning the four studied polymorphisms in some subjects did not reveal any difference in the nucleotide variance pattern, compared with the TNF-alpha reference sequence. Relative TNF-alpha mRNA expressions in HCV patients and healthy subjects were statistically indifferent.&#x0D; Conclusion: Since previous studies confirmed an increase in TNF-alpha level in case of viral infections, this study focuses on mechanisms of post-transcriptional and posttranslational modifications of TNF-alpha gene in HCV patients, which decode the genetic factors linked to HCV infection and severity.&#x0D;

Dietary soybean β‐conglycinin suppresses growth performance and inconsistently triggers apoptosis in the intestine of juvenile grass carp ( Ctenopharyngodon idella ) in association with ROS‐mediated MAPK signalling

The current study aimed to investigate the effects of dietary soybean beta-conglycinin on growth performance and intestine apoptosis in juvenile grass carp (Ctenopharyngodon idella). For fish fed with the 80 g beta-conglycinin/kg diet for 7 weeks, the specific growth rate and feed intake were decreased. In the proximal intestine, dietary beta-conglycinin did not induce DNA fragmentation, tended to decrease the reactive oxygen species (ROS) content, and decreased ROS-generating enzyme (NADPH oxidase [NOX]) activity. Subsequently, in the mid-intestine, dietary beta-conglycinin caused DNA fragmentation, tended to increase the ROS content, increased caspase-3, caspase-8 and caspase-9 activities, upregulated the mRNA levels of proapoptotic molecules (apoptotic protease-activating factor-1 [Apaf1] and Bcl-2-associated X protein [BAX]) and mitogen-activated protein kinase (MAPK)-related signal molecules (Jun N-terminal kinase (JNK) and p38 MAPK) and increased the protein levels of p38 MAPK and phospho-p38 MAPK. Moreover, in the distal intestine, dietary beta-conglycinin induced DNA fragmentation, elevated NOX activity and the ROS content and increased caspase-3, caspase-8 and caspase-9 activities, death ligand (TNF-alpha) mRNA expression level, and p38 MAPK and phospho-p38 MAPK protein levels. In summary, dietary soybean beta-conglycinin suppressed fish growth and inconsistently caused apoptosis among the different intestinal segments which was partially associated with ROS-mediated MAPK signalling.

OP-17 - Tea Extracts-induced Liver Injury: Lipotoxic Interaction Between Lipids and Polyphenols

Green tea polyphenol extracts (GTE) are sold commercially as cholesterol-reducing agents and for weight reduction however, they have also been reported to be hepatotoxic. Our results from both in vitro and in vivo studies indicate that green tea polyphenols can potentiate liver injury after and during treatment with high cholesterol diet to induce experimental NASH. Eight week old male C57BL mice (n=32) were fed for 6 weeks with one of the following diets: Control diet (Con); Con +1% w/w polyphenols from green tea extract (Con+GTE); High cholesterol diet, Con+ 1% cholesterol+ 0.5% cholate w/w (HCD); HCD + 1 % polyphenols w/w (HCD+GTE). Hepatic steatosis, oxidative and inflammatory markers and bile acid synthesis pathways were measured. GTE enhanced hepatic steatosis but only in animals exposed to the high cholesterol diet. In HCD treated animals GTE elevated blood levels of liver enzymes SGOT, SGPT, and bile acids. Inflammatory and oxidative markers in the liver were also significantly increased including liver mRNA expression of TNF-alpha, IL-6, SAA1, SAA2, iNOS and levels of 4-hydroxynonenal protein adducts. The mechanism of action for GTE/cholesterol hepatotoxicity was found to be related to alteration in bile acids synthesis pathways. This study shows that liver injury in the presence of lipids could be the reason for weigh loss by high dose of polyphenols.

The role of endothelin-1 and endothelin receptor antagonists in allergic rhinitis inflammation: ovalbumin-induced rat model

Desloratadine is a biologically active metabolite of loratadine which is indicated for the treatment of allergic rhinitis. Bosentan is a dual endothelin receptor antagonist used to treatment of pulmonary artery hypertension (PAH). In this study, we aimed to determine the role of endothelins in allergic rhinitis (AR) and the effects of endothelin receptor antagonists in AR rat models through comparison with desloratadine. In total, 20 adult Sprague-Dawley rats were used in this study. An ovalbumin-induced allergic rhinitis model was formed in three study groups except for the control group. Bosentan (100 mg/kg/day) was given to the bosentan-treated group for 7 days and desloratadine (10 mg/kg/day) was administered to the antihistaminic-treated group for 7 days. Nasal symptom scorings and histopathological examinations of the nasal tissues were carried out. Serum IgE levels and ET-1 and TNF-alpha mRNA expression levels were analysed. Between group comparisons for nasal symptoms, histopathological analysis, and molecular analyses were performed with a one-way ANOVA and Duncans multiple comparison tests. Significance was accepted at p smaller than 0.05. Bosentan inhibited nasal symptom more significantly than desloratadine. The IgE level, ET-1 and TNF-alpha mRNA expression levels statistically increased in the allergic rhinitis group when compared to other groups. Conversely, the bosentan-treatment group showed a significant recovery from the same parameters. The deterioration in histopathological parameters reached the highest levels in the allergic rhinitis group. The histopathological findings were close to those of the control group in the bosentan and antihistaminic-treated group. ET-1 is one of the mediators that impact AR development and ET-1 antagonists can be useful for symptom control and for decreasing allergic inflammation in AR patients.