mRNA Extraction Research Articles

MRNA expression of pattern recognition receptors and their signaling mediators in healthy and diseased gingival tissues.

Background:Gingivitis and periodontitis are initiated by inflammation caused by microorganisms. Pathogen-associated molecular patterns (PAMPs) from these microorganisms are recognized through various toll-like receptors (TLRs) and NOD-like receptors (NLRs). In this study, we have chosen five TLRs and two NLRs as representatives taking part in the recognition and inflammation process, along with a few of their signaling mediators including CD14, MYD88, and TRIF to compare their mRNA expression levels between healthy and diseased gingival tissues. This will provide deeper insight into the mechanisms underlying gingivitis and periodontitis. Understanding the mechanisms involved in the onset and progression of the periodontal diseases could greatly help in establishing effective ways for prevention and treatment of these diseases besides decreasing the risk factor for relevant systemic disorders.Materials and Methods:Gingival tissue samples for mRNA extraction and cDNA synthesis were taken from patients with gingivitis and periodontitis and from healthy control subjects. Messenger RNA expression of all genes was assessed using real-time polymerase chain reaction (PCR).Results:Among the genes studied in different groups, only MYD88 mRNA expression was significantly higher in the periodontitis group compared to that of the controls. The expression level of this molecule was also significantly higher in patients with severe periodontitis compared to other patients and also compared to healthy individuals. In different tissues, positive significant correlations were observed between the mRNA expression levels of some genes.Conclusions:Elevated mRNA levels of MYD88 in periodontitis might have a key role in the pathogenesis of this disease. Therefore, MYD88 may be a useful target for the therapy of this disease.

Evidence for Tumour Suppressor Function of DOK7 in Human Breast Cancer

Introduction: Downstream of tyrosine kinase 7 (DOK-7) is a member of the DOK family, which has been associated with the development and progression of various humancancers. Previously, identification of CpG hypermethylation in DOK-7 promoter was identified in breast cancer. Method: DOK-7 mRNA extraction and reverse transcription were performed on fresh frozen breast cancer tissue samples and normal background breast tissue. Transcript levels of expression were analyzed against TNM stage, tumour grade and clinical outcome over a 10-year follow-up period. Results: Levels of DOK-7 expression decreased significantly with increasing TNM stage. Higher DOK-7 expression was correlated with longer disease free and overall survival times. Conclusion: To our knowledge, this is the first study to investigate DOK-7 expression in human breast cancer. We identify a potential DOK-7 tumour suppressor role. DOK-7 as a prognostic biomarker in human breast cancer should be included in future validation studies.

Expression of glutathione S-transferase variants in human airway wall after long-term response to sulfur mustard

Context: Sulfur mustard (SM) is an alkylating agent identified as a potent chemical warfare agent. More recently, SM was used in the Iraq conflict against Iranian troops and civilians. At present, there are many people suffering from chronic obstructive pulmonary disease (COPD) due to mustard gas in Iran. SM increases the endogenous production of reactive oxygen species (ROS). The oxidant/antioxidant imbalance present in the lungs of these patients also results from the impaired capacity of the antioxidant/detoxification enzymes to detoxify the harmful reactive oxygen metabolites. Objective: One of the major antioxidants in human airways is glutathione S-transferase. They facilitate the detoxification of various environmental of oxidative stress. In this study, we attempted to understand the significance different in expression of GSTs in airway wall of chemical patients and control. Materials and methods: Seven normal and 20 SM induced COPD individuals were studied. Bronchoscopy was performed in all subjects and two specimens were taken from the main bronchus for mRNA extraction, PCR analysis and immunohistochemistry. Results: SM-induced COPD individuals showed expression of GSTA1 2.51 ± 0.83-, GSTM1 2.84 ± 1.71- and GSTP1 5.61 ± 2.59-folds higher than those of controls that revealed. GSTP1-immunoreactivity was strongly expressed in luminal border of normal samples. SM patient samples immunoreactivity for GSTP1 in the same area were negative. Discussion and conclusion: According to these findings, we speculated that overexpression of GSTs mRNA in patients revealed that GSTs plays an important role in cellular protection against oxidative stress of MS in airway wall of patients.

Streamlining gene expression analysis: integration of co-culture and mRNA purification

Co-culture of multiple cell types within a single device enables the study of paracrine signaling events. However, extracting gene expression endpoints from co-culture experiments is laborious, due in part to pre-PCR processing of the sample (i.e., post-culture cell sorting and nucleic acid purification). Also, a significant loss of nucleic acid may occur during these steps, especially with microfluidic cell culture where lysate volumes are small and difficult to access. Here, we describe an integrated platform for performing microfluidic cell culture and extraction of mRNA for gene expression analysis. This platform was able to recover 30-fold more mRNA than a similar, non-integrated system. Additionally, using a breast cancer/bone marrow stroma co-culture, we recapitulated stromal-dependent, estrogen-independent growth of the breast cancer cells, coincident with transcriptional changes. We anticipate that this platform will be used for streamlined analysis of paracrine signaling events as well as for screening potential drugs and/or patient samples.

187 INCREASED EFFICIENCY OF DERIVING BOVINE STEM CELL-LIKE COLONIES USING VALPROIC ACID AND SMALL-MOLECULE COCKTAILS

Nonviral reprogramming of bovine embryonic and adult fibroblasts was undertaken using electroporation of a polycistronic plasmid carrying human reprogramming factors Oct4, Sox2, Lin28, and Nanog (Addgene Plasmid #20922). Because of difficulties encountered in reliably and reproducibly reprogramming bovine cells to a pluripotent state, several small-molecule combinations have been tested to support reprogramming. Previously, a combination of three molecules, (sodium butyrate, PD0325901 and SB431542; NaB-PD-SB) in induced pluripotent stem (iPS) medium [Minimum Essential Medium Alpha, 20% FBS, 1x insulin-transferrin-selenium (ITS), 2 mM Glutamax (Gibco, Grand Island, NY, USA), 100 μM nonessential amino acids (NEAA), 50 U mol–1 penicillin, 50 mg mL–1 streptomycin, 0.1 mM β-mercaptoethanol, 4 ng mL–1 human leukaemia inhibitory factor, and 10 ng mL–1 basic fibroblast growth factor] was found to accelerate the reprogramming process of bovine iPS cells, with colonies observed at 12 days post-electroporation, as opposed to 21 days without the addition of the small-molecule cocktail. The addition of 1 mM valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, in combination with the other small-molecule cocktail (NaB-PD-SB), was found to improve the reprogramming efficiency of bovine adult and embryonic fibroblasts, with colonies observed at Day 10 post-electroporation, and the average number of colonies present per 10-cm dish, rising from an average of 5 colonies observed in the NaB-PD-SB cocktail to 27 with the addition of VPA. Colonies grown in the presence of VPA had a consistent morphology, forming compact domed colonies consisting of small round cells with well-defined borders. It was observed that colonies growing in the presence of VPA tended to have a better defined border and grew larger in size than those grown in the presence of NaB-PD-SB alone. Colonies began to differentiate after 21 days and were no longer alkaline phosphatase positive after this time. Cells were either harvested for mRNA extraction or differentiated into embryoid bodies (EB). Expression of pluripotency genes, Oct4, Sox2, Klf4, Nanog, and Alkaline Phosphatase were significantly increased in the presence of VPA compared to nonreprogrammed somatic bovine fibroblasts, with expression profiles similar to those grown in the NaB-PD-SB cocktail. Embryoid bodies were analysed for gene expression of different germ layer markers, FOXA2 (endoderm), Nestin and TUBB3 (ectoderm), and Desmin (mesoderm) using quantitative RT–PCR. For both EBs derived from the NaB-PD-SB cocktail and those with VPA, at least one marker from each germ layer was present, demonstrating the potential potency of these cells. At least a 10-fold increase in expression of these was observed in comparison to somatic fibroblast cells. It is apparent that the addition of small molecules can assist in the reprogramming of bovine iPS cells, and addition of VPA to the cocktail results in more consistent putative bovine iPS colonies. Further work is needed to identify the causes for early differentiation of colonies in order to obtain fully reprogrammed pluripotent stem cells.

Corrections for mRNA extraction and sample normalization errors find increased mRNA levels may compensate for cancer haplo‐insufficiency

The relative mRNA levels of differentially expressed (DE) and housekeeping (HK) genes of six aneuploid cancer lines with large-scale genomic changes identified by SNP/SKY analysis were compared with similar genes in diploid cells. The aneuploid cancer lines had heterogeneous genomic landscapes with subdiploid, diploid, and supradiploid regions and higher overall gene copy numbers compared with diploid cells. The mRNA levels of the haploid, diploid, and triploid HK genes were found to be higher after correction of easily identifiable mRNA measurement errors. Surprisingly, diploid and aneuploid HK gene mRNA levels were the same by standard expression array analyses, despite the higher copy numbers of the cancer cell HK genes. This paradoxical result proved to be due to inaccurate inputs of true intra-cellular mRNAs for analysis. These errors were corrected by analyzing the expression intensities of DE and HK genes in mRNAs extracted from equal cell numbers (50:50) of intact cancer cell and lymphocyte mixtures. Correction for both mRNA extraction/sample normalization errors and total gene copy numbers found the SUIT-2 and PC-3 cell lines' cancer genes both had ∼50% higher mRNA levels per single allele than lymphocyte gene alleles. These increased mRNA levels for single transcribed cancer alleles may restore functional mRNA levels to cancer genes rendered haplo-insufficient by the genetic instability of cancer. © 2013 Wiley Periodicals, Inc.

Damage‐associated molecular patterns stimulate interleukin‐33 expression in nasal polyp epithelial cells

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a disorder characterized by eosinophilic inflammation and local T-helper 2 (Th2) cytokine production. Innate lymphoid cells that elaborate Th2 cytokines have recently been characterized within nasal polyps. These cells can be activated by the epithelial cell-derived cytokine interleukin-33 (IL-33). The objective of this study is to determine whether 2 molecules associated with tissue damage (high mobility group box-1 [HMGB-1] and adenosine triphosphate [ATP]) elicit expression of IL-33 in sinonasal epithelial cells (SNECs) derived from recalcitrant CRSwNP patients. Ethmoid tissue was obtained from 8 recalcitrant CRSwNP and 9 control subjects during endoscopic sinus surgery (ESS). Tissue was prepared for immunohistochemistry and for SNEC air-liquid interface culture. After exposure to either HMGB1 or ATP in vitro, SNECs were processed for messenger RNA (mRNA) extraction and immunocytochemistry. IL-33 levels were determined by real-time polymerase chain reaction (PCR) and by immunochemical staining with anti-IL-33 antibody. Intranuclear IL-33 is normally expressed in basal epithelial cells, but is present in more apical cells and outside the nucleus in CRSwNP. Exposure of SNECs to HMGB-1 or ATP resulted in a statistically significant increase in IL-33 mRNA expression in SNECs derived from recalcitrant CRSwNP patients. This increase was reflected at the protein level by immunochemical staining of IL-33. Tissue damage is a nonspecific trigger of epithelial IL-33 production in treatment-recalcitrant polyps, which may be responsible for perpetuating eosinophilic inflammation in CRSwNP. This common pathway may help explain why multiple environmental and infectious agents have been implicated in CRSwNP exacerbation.

98-P: KIR mRNA EXPRESSION IN PBMC AND ENRICHED NK CELLS ASSAYED BY QUANTITATIVE REAL-TIME PCR

To develop assays to quantify the mRNA transcribed from KIR genes utilizing a quantitative real-time PCR (qPCR) method. To study the KIR mRNA expression patterns in PBMC and enriched NK cells among healthy volunteers. PBMCs were obtained from freshly drawn whole blood (maximum 48 hours) from 14 healthy volunteers using a Ficoll method. NK cell enrichment from PBMC was performed by negative selection using a magnetic bead kit. mRNA extraction and cDNA synthesis was followed by EvaGreen based qPCR. Relative KIR mRNA expression was quantified by 2-ΔΔCt algorithm with GAPDH as reference. qPCR with a Cq ⩽ 38.0 was considered positive. 10 mRNA expression assays for the 9 KIR genes 2DL1, 2DL5A, 2DL5B, 2DS1, 2DS2, 2DS3, 2DS4 (2DS4-full length and 2DS4-truncated), 2DS5 and 3DS1 have been developed and validated. Among the 14 individuals, KIR mRNAs were detected in all samples carrying respective KIR genes and were negative for KIR gene negative samples. The mRNA expression varied among individuals. In PBMC, the relative quantification (RQ) of cDNA showed the largest variation for 2DL1 (14-fold, n = 14) and the smallest for 3DS1 (1.8-fold, n = 3) among volunteers who were positive for the respective genes. 2DL5B cDNA was not detected, as expected for this gene. More KIR cDNA was detected in the enriched NK cell fraction when compared to PBMC, with the largest difference observed for 2DL1 (NK : PBMC ratio, mean ± SD: 37.8 ± 25.0, n = 14). Development of qPCR assays with high specificity and easy applicability will be useful, because all known 15 KIR genes share > 90% identity at the genomic level. We established 10 assays for the mRNA expression of 9 KIR genes. KIR mRNA expression varied among healthy individuals carrying the same KIR genotype. Because NK cells are the predominant, but not the only source of KIR mRNA, analyzing PBMC and NK cells will allow establishing an overall and NK cell specific KIR mRNA expression signature from an individual.

Keratinocyte growth factor, tumor necrosis factor-alpha and interleukin-1 beta gene expression in cultured fibroblasts and keratinocytes from burned patients

To evaluate the gene expression of KGF, TNF-alpha and IL-1 beta in skin fibroblasts and keratinocytes cultured from burned patients. Three patients with large burns and three patients with small burns, as well as two controls, were included. The cell culture was initiated by the enzymatic method. After extraction and purification of mRNA, qPCR was used to assess the gene expression of KGF, TNF-alpha and IL-1 beta. The expression of KGF was increased on average 220-fold in large burns and 33.33-fold in small burns in fibroblasts, and 11.2-fold in large burns and 3.45-fold in small burns in keratinocytes compared to healthy patients (p<0.05). Expression of TNF-alpha was not observed. IL-1 beta is down-regulated in fibroblasts of burned patients, and much more repressed in small burns (687-fold, p<0.05). In keratinocytes, the repression of IL-1 beta expression occurs in patients with small burns (28-fold), while patients with large burns express this gene intensively (15-fold). The study showed a quantitative pattern in the expression of KGF gene, which is more expressed according to the size of the burn. TNF-alpha was not expressed. A qualitative pattern in the expression of IL-1 beta gene was demonstrated.

Msx2 plays a critical role in lens epithelium cell cycle control.

To investigate the effects of Msx2 on lens epithelium cell cycle, and evaluate the changes of the proliferation, apoptosis of lens epithelium cells. Mice lens epithelium cells were cultured and transfected with pEGFP-Msx2 and control. Msx2-deficient mice (Msx2(-/-) ) lens tissue were isolated. Lens tissue and transfected cells were prepared for mRNA extraction using Trizol reagent. CyclinD1 and Prox1 expression were evaluated by real-time RT-PCR. BrdU incorporation and apoptosis rate were investigated by immunofluorescence and flow cytometry analysis. After transfected with pEGFP-Msx2, lens epithelium cells failed to incorporate BrdU and anti-phospho-histone-3 immunofluorescence failed to detect cell nuclei which GFP were positive. Msx2 over expression resulted in increasing apoptosis rate in lens epithelium cells. CyclinD1 and Prox1 expression increased significantly in Msx2 knockout mice by real-time RT-PCR quantization and CyclinD1 expression decreased significantly in Msx2 overexpressed cell. Msx2 has the effect of inhibiting proliferation and differentiation, triggering apoptosis on mice lens epithelium cells.

Adipose Tissue Collagen and Inflammation in Nonobese Asian Indian Men

Obesity is thought to be the driving force for activation of adipose tissue (AT) collagen production and inflammation as well as systemic insulin resistance. The objective of this study was to determine whether these AT abnormalities can be found independent of obesity in the presence of systemic insulin resistance. Thirty-eight normoglycemic men (14 Asian Indians and 24 white) were enrolled in the study and matched for age, body mass index, and total body fat. Subjects underwent anthropometric measurement, total body fat determination by underwater weighing, euglycemic-hyperinsulinemic clamps, and abdominal sc AT biopsy for mRNA extraction and gene expression determination. Fasting blood was collected for adipokine measurements. Both groups were matched for age, body mass index, and percentage of total body fat. Subcutaneous abdominal AT mRNA expression was significantly higher for Col6a3 as well as genes associated with inflammation, CD68, MAC1, and MCP1 in Asian Indians compared with whites. Asian Indian men had significantly lower rates of glucose disposal and lower plasma adiponectin concentration. Plasma high-sensitivity C-reactive protein levels showed a trend towards higher levels in Asian Indian men. Increased col6a3 and macrophage infiltration in AT along with increased systemic insulin resistance is present independent of body fat content in young Asian Indian men, thus suggesting that AT dysfunction associates with systemic insulin resistance regardless of AT mass.

An alternative method for producing Toxocara canis second stage larvae from a paratenic host (pigeon) for mRNA extraction purpose.

Toxocara canis is a prevalent zoonotic parasite which can cause serious disease in puppies and humans. Excretory-secretory and coating antigens of the second stage larvae (L2) are the best targets for performing immunodiagnostic and also immunoprophylactic tests. Various hatching methods have been described to bring out L2 from the resistant infective egg shell; but these methods are difficult to do and have had different results when performed by different practitioners. In this study, second stage larvae were obtained from the viscera of pigeons (a paratenic host) which were infected with infective eggs. Infective Toxocara canis eggs were given to ten pigeons and live larvae were recovered from their excised livers and lungs by using the Baermann's apparatus in the next days. Two in vitro methods for larvae hatching were also performed including a so-called physiological hatching method according to Ponce-Macotela et al. (J Parasitol 175:382-385, 2010), and a mechanical hatching method according to Alcântara-Neves and Santos (J Exp Parasitol 119:349-351, 2008) and their results were compared with the in-vivo method. Results show that averagely 36.2% of fed larvae recovered from livers and 0.15% from lungs. Average larvae recovery in the first day after infection (24.2%) was significantly lower than subsequent days (39%). Maximum larvae recovered in day 3 (55%). In-vitro methods we carried out did not have acceptable results and only a few larvae did hatch using these methods.

Assessment of the viability of Cryptosporidium parvum oocysts with the induction ratio of hsp70 mRNA production in manure

Determining the presence of viable Cryptosporidium parvum oocysts in complex environmental matrices in hygiene control can prevent the contamination of water resources and food with this pathogen. This study assessed the induction ratio of hsp70 mRNA production by heat shock in different oocysts as a marker of viability. Using different procedures for (m)RNA extraction directly from manure and reverse transcription real-time qPCR, this study found slightly increased hsp70 mRNA contents in viable oocysts that were heat shock induced at 45°C for 20min compared to not induced oocysts (1.6 fold induction in average). Prolonging the heat shock treatment to 2h did not further increase the copy numbers. Heat shock by consecutive stimuli, such as freezing and then heating, did not yield significantly higher copy numbers than the 45°C treatment. There was a certain background level of hsp70 mRNA in viable oocysts that were not exposed to heat shock, indicating a constitutive production of the transcripts in the oocysts. The production of hsp70 mRNA induced by heat shock in oocysts aged for 9months that exhibited reduced viability was lower than in fresher oocysts (induction ratio<1.2). No production of hsp70 mRNA by heat shock was detected in 12months old oocysts that were not viable in the excystation test. Oocysts inactivated at 75°C for 30min were not able to respond to heat shock, and low amount of copies were occasionally measured only in total RNA extracts, but not in mRNA extracts that were purified directly with an oligo (dT)25 based system. The induction ratio of hsp70 mRNA varied according to the viability of the organisms in a sample. Copy numbers of β-tubulin mRNA in viable oocysts were lower than hsp70 mRNA, therefore the latter is more suitable to detect low numbers of oocysts by RT-qPCR.

PTH-087 Fam5C, a Possible Causal Molecule in Ulcerative Colitis Revealed through a Transcriptomic Analysis of the Bowel Mucosa

IntroductionAbnormalities of the colonic mucosa have been implicated in the pathogenesis of ulcerative colitis (UC). We investigated mRNA profiles of macroscopically non-inflamed mucosal biopsies from the colon in patients with...

AB0096 NF-KB involvement for CTLA4-IG intracellular signaling in human macrophages

BackgroundTranscription factor NF-kB (nuclear factor kB) play an important role in the regulation of immune and inflammatory responses [1]. Previous studies showed that a significant downregulation of pro-inflammatory cytokine expression...

Su1770 Fam5c, a Possible Causal Molecule in Ulcerative Colitis Revealed Through a Transcriptomic Analysis of the Bowel Mucosa

Introduction Abnormalities of the colonic mucosa have been implicated in the pathogenesis of ulcerative colitis (UC). We investigated mRNA profiles of macroscopically non-inflamed mucosal biopsies from the colon in patients with UC, Crohn’s disease (CD) and control subjects without gastrointestinal disease (HC), to identify genes that might be involved in the aetiology of the disease. Methods Paired biopsies were taken for histology and mRNA extraction from macroscopically non-inflamed mucosa in the ascending and descending colon, and the rectum, from 24 patients with UC, 14 with CD and 27 HCs undergoing routine colonoscopy. Patients were in complete clinical remission and were either on no treatment or on 5-aminosalicylates ± azathioprine. cRNA was hybridised to Illumina HumanHT-12 v4 Expression Beadchips. Array expression data were log transformed and normalised. Only probes with a detection p-value Results In group comparisons, of the 26,261 expressed probes, Family with Sequence Similarity 5, member C ( FAM5C ) was the only gene to be significantly under-expressed in UC, both in the rectum (FC = –1.58, p = 0.0008) and the descending colon (FC = –1.64, p = 0.0011). Outlier analysis showed that FAM5C was also grossly under-expressed in the ascending colon in 37.5% of UC patients, demonstrating that its expression is abnormal throughout the colon in a significant proportion of individuals. Expression levels were not abnormal in CD. Expression of FAM5C in UC did not correlate with the known markers of inflammation, IL-8 , S100A8 , DEFA5 and DEFA6 , or with treatment. The under-expression of FAM5C in UC was confirmed in biopsies of non-inflamed rectal mucosa from an independent cohort of patients (FC = –1.68, p = 0.0073) and by qPCR (p Conclusion This is the first description of the under-expression of FAM5C in UC. As these observations were made in non-inflamed mucosa, low levels of this protein might be involved in the pathogenesis of the disease. Indications that FAM5C may function as tumour suppressor [1], could link to the observed predisposition to colonic malignancy in UC. Disclosure of Interest None Declared. Reference Kuriowa T et al. (2009) Oncol Rep, 1005–11.

Inhibition of Hypoxia Inducible Factor (HIF)-1α Suppresses Formation and Progression of Experimental Abdominal Aortic Aneurysms (AAAs)

elevations of serum creatinine occur days later and research into makers of early kidney injury has focused on various proteins with disappointing results. Our animal data revealed that even low dose contrast leads to kidney injury and nephron-specific mRNA can be detected in the urine. We wished to evaluate if nephron-specific mRNA could be detected in patients after endovascular procedures as a new, specific marker for CIKI. Methods: Adults undergoing endovascular procedures requiring contrast were consented. Urine was collected over 24hrs. Urinalysis and mRNA extraction followed. rtPCR using nephron-specific (Nephrin, Podocin, Aquaporin-2) and a non-specific (TBF-Beta) probes. Clinical data was also collected. Results: Avg contrast:116cc. Avg age:69. Significant increases were found only in Nephrin mRNA at 6hrs, and returned to baseline (n 1⁄4 20; P 1⁄4 .03 see graph). Hematuria preceded this at 3hrs (P < .02) after contrast exposure. There was no increase in the serum Cr (it decreased). Conclusions: Consistent with our animal model, Nephrin mRNA and blood are detectable in the urine within hours after contrast exposure, regardless of serum creatinine. Urine mRNA is a potentially sensitive marker for CIN.

The role of Toll-like receptor proteins (TLR) 2 and 4 in mediating inflammation in proximal tubules

Inflammatory responses are central to the pathogenesis of diabetic nephropathy. Toll-like receptors (TLRs) are ligand-activated membrane-bound receptors which induce inflammatory responses predominantly through the activation of NF-κB. TLR2 and 4 are present in proximal tubular cells and are activated by endogenous ligands upregulated in diabetic nephropathy, including high-mobility group box-1 (HMGB1) and fibronectin. Human proximal tubules were exposed to 5 mM (control), 11.2 mM (approximating the clinical diagnostic threshold for diabetes mellitus), and 30 mM (high) glucose for 72 h or 7 days. Cells were harvested for protein, mRNA, and nuclear extract to assess for TLR2, 4, and inflammatory markers. Glucose (11.2 mM) maximally increased TLR2 and 4 expression, HMGB1 release, and NF-κB activation with increased expression of cytokines. However, only TLR2 expression and subsequent NF-κB binding were sustained at 7 days. Recombinant HMGB1 induced NF-κB activation, which was prevented by both TLR2 silencing [small interfering (si)RNA] and TLR4 inhibition. Peroxisome proliferator-activated receptor-γ (PPAR-γ) transcription was reduced by exposure to 11.2 mM glucose with an increase observed at 30 mM glucose at 24 h. This may reflect a compensatory increase in PPAR-γ induced by exposure to 30 mM glucose, limiting the inflammatory response. Therefore, short-term moderate increases in glucose in vitro increase HMGB1, which mediates NF-κB activation through both TLR2 and 4. Furthermore, in vivo, streptozotocin-induced diabetic mice exhibited an increase in tubular TLR2 and HMGB1 expression. These results collectively suggest that TLR2 is likely to be the predominant long-term mediator of NF-κB activation in transducing inflammation in diabetic nephropathy.

PP029. Genetic change of adipose tissue related to inflammation in preeclampsia: Findings under novel culture method

Recently, adipose tissue is suggested to contribute to the inflammatory action in preeclampsia(PE), from peripheral increase of cytokines. However, the mechanism of the action in adipose tissue was not clarified yet. In this study, we performed "Gel bottom-captured" adipose tissue culture with PE to show that the adipose tissue is the inflammatory focus in the pathophysiology of PE. Under informed consent of the patients, omentum from probe laparotomy for ovarian cancer was captured in Peptide Hydrogel®. After 24h starvation, tissues were cultured with medium containing 10% of severe PE and healthy pregnant maternal serum (n=5 each). M30 (Apoptosis) and M65 (all cell death) were measured with ELISA. After mRNA extraction from tissue, quantitative PCR array (Qiagen, Inc.) was performed on all samples. No significant histological differences were found between each culture. However, M30/M65 ratio was increased in PE (p=0.053). In PCR array, under 2-fold decrease in OSM (p=0.019) was found, and over 2-fold increase in TLR (p<0.01) and other inflammatory genes were defined in PE. Inflammatory action in PE via TLR pathway was defined by adipose tissue culture. Apoptosis were shown in PE condition, but total tissue injury include necrosis were suggested to be stronger in normal pregnancy. Increase of inflammatory gene suggested that adipose tissue is one of a main organ of systemic inflammation in PE.

The Impact of High Mobility Group Box 1 Protein (HMGB1) on Renal Ischemia‐Reperfusion Injury in Male and Female Spontaneously Hypertensive Rats (SHR)

HMGB1 is an endogenous molecule released by dying cells which contributes to renal ischemia‐reperfusion injury (IRI). We recently published that male SHR have more pro‐inflammatory immune cells in the kidney. The goal of this study was to test the hypothesis that female SHR have less HMGB1 than males resulting in a lower degree of renal IRI. HMGB1 levels were measured via ELISA in kidneys of 12 week old male and female SHR (N=5). Additional age matched rats were pre‐treated with control or HMGB1 neutralizing antibody (300 μg/rat via IP; N=4) 1 hour before occluding renal arteries for 1 hour. Renal blood flow velocity (RBV) was measured via ultrasound before and 24 hours after reperfusion. Kidneys were isolated for mRNA extraction and histology. HMGB1 levels were greater in the renal cortex of female vs. male SHR (0.33±0.05 vs. 0.16±0.02 ng/mg; p=0.01). Medullary HMGB1 levels were comparable between sexes. Pre‐treatment with anti‐HMGB1 did not alter RBV compared to controls (% decrease of male SHR: 34±2 to 35±5, NS; female SHR: 35±6 to 22±6, NS) or expression of renal VCAM1 or ICAM1 in either sex. Using histological analysis we determined that anti‐HMGB1 decreased CD3+ T cell infiltration in both sexes, verifying a role for HMGB1 in inflammation following IRI. In conclusion, despite greater renal HMGB1 levels in female SHR, there were no sex differences in the functional role of HMGB1 inhibition on renal IRI or inflammation.