Cyclooxygenase-2 Positive Cells Research Articles

Disaggregation-Activated pan-COX Imaging Agents for Human Soft tissue Sarcoma.

Cancer stem cells are pivotal players in tumors initiation, growth, and metastasis. While several markers have been identified, there remain challenges particularly in heterogeneous malignancies like adult soft tissue sarcomas, where conventional markers are inherently overexpressed. Here, we designed BODIPY scaffold fluorescence probes (BD-IMC-1, BD-IMC-2) that activate via disaggregation targeting for cyclooxygenase (COX), a potential marker for CSCs in sarcoma in clinical pathology. Based on their structures, BD-IMC-1 showcased higher susceptibility to disaggregation compared to BD-IMC-2, consistent with their selective interaction with COX. Notably, the BD-IMC-1 revealed positive cooperativity binding to COX-2 at sub-micromolar ranges. Both probes showed significant fluorescence turn-on upon LPS or PMA triggered COX-2 upregulation in live RAW264.7, HeLa, and human sarcoma cell line (Saos-LM2) up to 2-fold increase with negligible toxicity. More importantly, the BD-IMC-1 demonstrated their practical imaging for COX-2 positive cells in paraffin-fixed human sarcoma tissue. Considering the fixed tissues are most practiced pathological sample, our finding suggests a potential of disaggregation activated chemosensor for clinical applications.

Firocoxib as a Potential Neoadjuvant Treatment in Canine Patients with Triple-Negative Mammary Gland Tumors

This study aimed to investigate the pro-apoptotic effects of NSAID (Previcox®) in vitro and in vivo. Two CMT cell lines, one from the primary tumor and one from bone metastasis, were treated with firocoxib and MTT assay was performed to determine the half-maximal inhibitory concentration (IC50) value. The firocoxib IC50 for the cell lines UNESP-CM5 and UNESP-MM1 were 25.21 µM and 27.41 µM, respectively. The cell lines were then treated with the respective firocoxib IC50 concentrations and annexin V/propidium iodide (PI) assay was performed, to detect the induction of apoptosis in both cells (Annexin+/PI+). We conducted an in vivo study involving female dogs affected by CMT and divided them into control and treatment groups. For both groups, a biopsy was performed on day 0 (D0) and a mastectomy was performed on day 14 (D14). In the treatment group, after biopsy on D0, the patients received Previcox® 5 mg/kg PO once a day until mastectomy was performed on D14. COX-2/caspase-3 double immunostaining was performed on samples from D0 and D14, revealing no difference in the control group. In contrast, in the treatment group Previcox® increased the number of COX-2 positive apoptotic cells. Therefore, firocoxib can induce apoptosis in CMT cells in vitro and in vivo, and Previcox® can be a potential neoadjuvant treatment for patients with mammary cancer.

Ischemia-induced inflammation in arteriovenous malformations.

The pathophysiology of development, growth, and rupture of arteriovenous malformations (AVMs) is only partially understood. However, inflammation is known to play an essential role in many vascular diseases. This feasibility study was conducted to investigate the expression of enzymes (cyclooxygenase 2 [COX-2] and NLRP3 [NOD-, LRR-, and pyrin domain-containing protein 3]) in the AVM nidus that are essential in their inflammatory pathways and to explore how these influence the pathophysiology of AVMs. The study group comprised 21 patients with partially thrombosed AVMs. The cohort included 8 ruptured and 13 unruptured AVMs, which had all been treated microsurgically. The formaldehyde-fixed and paraffin-embedded samples were immunohistochemically stained with a monoclonal antibody against COX-2 and NLRP3 (COX-2 clone: CX-294; NLRP3: ab214185). The authors correlated MRI and clinical data with immunohistochemistry, using the Trainable Weka Segmentation algorithm for analysis. The median AVM volume was 2240 mm3. The proportion of NLRP3-positive cells was significantly higher (26.23%-83.95%), compared to COX-2 positive cells (0.25%-14.94%, p < 0.0001). Ruptured AVMs had no higher expression of NLRP3 (p = 0.39) or COX-2 (p = 0.44), compared to nonruptured AVMs. Moreover, no patient characteristics could be reported that showed significant correlations to the enzyme expression. NLRP3 consistently showed an approximately 10-fold higher expression level than COX-2, making the inflammatory process in AVMs appear to be mainly associated with ischemic (NLRP3)-driven rather than with mechanical (COX-2)-driven inflammatory pathways. No direct associations between NLRP3 and COX-2 expression and radiological, standard histopathological, or patient characteristics were found in this cohort.

Regulatory T Cells with Additional COX-2 Expression Are Independent Negative Prognosticators for Vulvar Cancer Patients.

Vulvar cancer incidence numbers have been steadily rising over the past decades. In particular, the number of young patients with vulvar cancer has recently increased. Therefore, the need to identify new prognostic factors and, in addition, therapeutic options for vulvar carcinoma is more apparent. The aim of this study was to analyze the influx of COX-2 positive tumor-infiltrating lymphocytes and monocytes and their influence on prognosis. Using subtyping by immunofluorescence, the majority of COX-2 expressing immune cells were identified as FOXP3-positive regulatory T cells. In addition, peri- and intra-tumoral macrophages in the same tumor tissue were detected simultaneously as M2-polarized macrophages. COX-2 positive immune cells were independent negative prognostic markers in long-term overall survival of patients with vulvar cancer. These results show an influence of immune cell infiltration for vulvar carcinoma patients. Immune cell infiltration and immune checkpoint expression may, therefore, become interesting targets for further research on new vulvar cancer treatment strategies.

The Upregulation of COX2 in Human Degenerated Nucleus Pulposus: The Association of Inflammation with Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IVDD) is an important risk factor of low back pain. We previously found upregulated markers of fibrosis, the late stage of chronic inflammation, in degenerated IVD with a small number of clinical specimens. Here, we aimed to study on a larger scale the association of cyclooxygenase 2 (COX2), an inflammation and/or pain marker, with IVDD. This study involved 107 LBP participants. The IVD degeneration level was graded on a 1–5 scale according to the Pfirrmann classification system. Discs at grades 1-3 were further grouped as white discs with grades 4-5 as black discs. We recorded baseline information about age, gender, body mass index (BMI), diabetes history, smoking history, and magnetic resonance imaging (MRI). Their association with IVDD was statistically analyzed. The expression level of COX2 was investigated by immunohistochemistry. The total integrated COX2 optical density (IOD), number of COX2-positive cells, and total cell number of each image were counted and analyzed by Image-Pro Plus software. The IOD and number of COX2-positive cells were divided by the total cell number to obtain COX2 expression density (IOD/cell) and COX2 positivity (cell+/cell). As a result, among the baseline information investigated, only age was found to have a significant association with IVDD. The IOD/cell was found to be significantly increased from grade 2 to grade 5, as well as in black discs compared to white discs. The cell+/cell displayed the same trend that it increased in highly degenerative discs compared to their counterparts. In conclusion, the expression of COX2 is associated with IVDD, which highlights COX2 as a biomarker for IVD degeneration and indicates the involvement of inflammation and pain signaling in IVDD.

Chronic intestinal inflammation drives colorectal tumor formation triggered by dietary heme iron in vivo

The consumption of red meat is associated with an increased risk for colorectal cancer (CRC). Multiple lines of evidence suggest that heme iron as abundant constituent of red meat is responsible for its carcinogenic potential. However, the underlying mechanisms are not fully understood and particularly the role of intestinal inflammation has not been investigated. To address this important issue, we analyzed the impact of heme iron (0.25 µmol/g diet) on the intestinal microbiota, gut inflammation and colorectal tumor formation in mice. An iron-balanced diet with ferric citrate (0.25 µmol/g diet) was used as reference. 16S rRNA sequencing revealed that dietary heme reduced α-diversity and caused a persistent intestinal dysbiosis, with a continuous increase in gram-negative Proteobacteria. This was linked to chronic gut inflammation and hyperproliferation of the intestinal epithelium as attested by mini-endoscopy, histopathology and immunohistochemistry. Dietary heme triggered the infiltration of myeloid cells into colorectal mucosa with an increased level of COX-2 positive cells. Furthermore, flow cytometry-based phenotyping demonstrated an increased number of T cells and B cells in the lamina propria following heme intake, while γδ-T cells were reduced in the intraepithelial compartment. Dietary heme iron catalyzed formation of fecal N-nitroso compounds and was genotoxic in intestinal epithelial cells, yet suppressed intestinal apoptosis as evidenced by confocal microscopy and western blot analysis. Finally, a chemically induced CRC mouse model showed persistent intestinal dysbiosis, chronic gut inflammation and increased colorectal tumorigenesis following heme iron intake. Altogether, this study unveiled intestinal inflammation as important driver in heme iron-associated colorectal carcinogenesis.

Urinary prostaglandin E2 as a biomarker for recurrent UTI in postmenopausal women.

Urinary tract infection (UTI) is one of the most common adult bacterial infections and exhibits high recurrence rates, especially in postmenopausal women. Studies in mouse models suggest that cyclooxygenase-2 (COX-2)-mediated inflammation sensitizes the bladder to recurrent UTI (rUTI). However, COX-2-mediated inflammation has not been robustly studied in human rUTI. We used human cohorts to assess urothelial COX-2 production and evaluate its product, PGE2, as a biomarker for rUTI in postmenopausal women. We found that the percentage of COX-2-positive cells was elevated in inflamed versus uninflamed bladder regions. We analyzed the performance of urinary PGE2 as a biomarker for rUTI in a controlled cohort of 92 postmenopausal women and PGE2 consistently outperformed all other tested clinical variables as a predictor of rUTI status. Furthermore, time-to-relapse analysis indicated that the risk of rUTI relapse was 3.6 times higher in women with above median urinary PGE2 levels than with below median levels. Taken together, these data suggest that urinary PGE2 may be a clinically useful diagnostic and prognostic biomarker for rUTI in postmenopausal women.

Histopathological and Immunohistochemical Study of the Distinction between Oral Lichen Planus and Oral Lichenoid Lesions

Background: Oral potentially malignant disorders, which include oral lichen planus (OLP), are clinical presentations that carry a risk of development to cancer in the oral cavity. Oral lichenoid lesions (OLLs) are also termed interface/lichenoid mucositis. Malignant transformation of them remains controversial, but distinct clinical and histological criteria for how to differentiate OLP from OLLs have not been developed. Objectives: The purpose of this study was to elucidate findings that can allow histopathological differentiation of OLP and OLLs using histomorphological and immunohistochemical analyses. Materials and Methods: Analyses were performed in 10 cases diagnosed with OLP and 9 cases diagnosed with OLLs. Cytokeratin 19 (CK19), Ki-67 and CD3 were used as primary antibodies to detect basal cells, proliferative activity and T-cell distribution, respectively, and Perlecan and COX-2 to evaluate epithelial intracellular arrangements and interstitial distributions of proteoglycans and enzymes. Results: For CK19, positive cells were significantly found in OLLs at both the prominent area and site adjacent to the lesion comparison with those of OLP’s. The number of COX-2 positive cells was significantly higher in spinous and basal layers in OLLs of the prominent area. Additionally, OLLs showed mild to moderate expression for perlecan in the basal to spinous layers and in subepithelial tissue. Conclusion: Almost no basal cells were noted in the prominent area in OLP. COX-2 and perlecan were found in the basal to spinous layers in OLLs. Although there are restrictions, these suggested the possibility of helping to distinguish between OLP and OLLs.

Dietary ARA Improves COX Activity in Broodstock and Offspring Survival Fitness of a Model Organism (Medaka Oryzias latipes).

Simple SummaryThe impact of arachidonic acid in broodstock diets on cyclooxygenase activity and quality of offspring was investigated on model fish species. The results have shown that arachidonic acid in the broodstock diet improves sperm motility parameters. The cyclooxygenase activity in the liver of females increased with the amounts of arachidonic acid in diets. The arachidonic acid bioconversion activity during the reproductive season via cyclooxygenase activity is involved in female fertility and embryo and larval survival and growth.A 3-week feeding trial was conducted in medaka broodstock (age five months) to examine the effect of dietary arachidonic acid (ARA) level (range: 4–23 mg g−1 of total fatty acids (TFAs)) on fertility, cyclooxygenase (COX) activity, egg size, sperm motility parameters, hatching rate and weight of hatch, survival and growth of larvae. After spawn induction and dietary exposure to 4 mg ARA g−1 TFA, broodstock were fed a diet containing ARA in the amounts: 4 (continued, as control), 5, 14 and 23 mg g−1 TFA. COX1 activity in the liver and the number of COX2-positive cells in the ovaries was increased in females fed the diets containing the two highest amounts of ARA. The highest sperm motility parameters were observed in males fed a diet containing 23 mg ARA g−1 TFA. The hatchability rate and bodyweight of hatchlings were higher in the group obtained from broodstock fed a diet containing 23 mg ARA g−1 TFA (79% and 0.66 mg fish−1, respectively) compared with 4 mg ARA g−1 TFA (50% and 0.40 mg fish−1). The average mortality of offspring obtained from this group at 7 days post hatching was significantly higher than that of all other groups.

Pathophysiology of Intracranial Aneurysms: COX-2 Expression, Iron Deposition in Aneurysm Wall, and Correlation With Magnetic Resonance Imaging.

The pathophysiology of development, growth, and rupture of intracranial aneurysms (IAs) is only partly understood. Cyclooxygenase 2 (COX-2) converts arachidonic acid to prostaglandin H2, which, in turn, is isomerized to prostaglandin E2. In the human body, COX-2 plays an essential role in inflammatory pathways. This explorative study aimed to investigate COX-2 expression in the wall of IAs and its correlation to image features in clinical (1.0T, 1.5T, and 3.0T) magnetic resonance imaging (MRI) and ultra-high-field 7T MRI. The study group comprised 40 patients with partly thrombosed saccular IAs. The cohort included 17 ruptured- and 24 unruptured IAs, which had all been treated microsurgically. Formaldehyde-fixed paraffin-embedded samples were immunohistochemically stained with a monoclonal antibody against COX-2 (Dako, Santa Clara, CA; Clone: CX-294). We correlated Perls Prussian blue staining, MRI, and clinical data with immunohistochemistry, analyzed using the Trainable Weka Segmentation algorithm. Aneurysm dome size ranged between 2 and 67 mm. The proportion of COX-2 positive cells ranged between 3.54% to 85.09%. An upregulated COX-2 expression correlated with increasing IA dome size (P=0.047). Furthermore, there was a tendency of higher COX-2 expression in most ruptured IAs (P=0.064). At all field strengths, MRI shows wall hypointensities due to iron deposition correlating with COX-2 expression (P=0.022). Iron deposition and COX-2 expression in IAs walls correlate with signal hypointensity in MRI, which might, therefore, serve as a biomarker for IA instability. Furthermore, as COX-2 was also expressed in small unruptured IAs, it could be a potential target for specific medical treatment.

Radiosynthesis and evaluation of [18F]FMTP, a COX-2 PET ligand.

The upregulation of cyclooxygenase-2 (COX-2) is involved in neuroinflammation associated with many neurological diseases as well as cancers of the brain. Outside the brain, inflammation and COX-2 induction contribute to the pathogenesis of pain, arthritis, acute allograft rejection, and in response to infections, tumors, autoimmune disorders, and injuries. Herein, we report the radiochemical synthesis and evaluation of [18F]6-fluoro-2-(4-(methylsulfonyl)phenyl)-N-(thiophen-2-ylmethyl)pyrimidin-4-amine ([18F]FMTP), a high-affinity COX-2 inhibitor, by cell uptake and PET imaging studies. The radiochemical synthesis of [18F]FMTP was optimized using chlorine to fluorine displacement method, by reacting [18F]fluoride/K222/K2CO3 with the precursor molecule. Cellular uptake studies of [18F]FMTP was performed in COX-2 positive BxPC3 and COX-2 negative PANC-1 cell lines with unlabeled FMTP as well as celecoxib to define specific binding agents. Dynamic microPET image acquisitionwas performed in anesthetized nude mice (n = 3), lipopolysaccharide (LPS) induced neuroinflammation mice (n = 4), and phosphate-buffered saline (PBS) administered control mice (n = 4) using a Trifoil microPET/CT for a scan period of 60min. A twofold higher binding of [18F]FMTP was found in COX-2 positive BxPC3 cells compared with COX-2 negative PANC-1 cells. The radioligand did not show specific binding to COX-2 negative PANC-1 cells. MicroPET imaging in wild-type mice indicated blood-brain barrier (BBB) penetration and fast washout of [18F]FMTP in the brain, likely due to the low constitutive COX-2 expression in the normal brain. In contrast, a ~ twofold higher uptake of the radioligand was found in LPS-induced mice brain than PBS treated control mice. Specific binding to COX-2 in BxPC3 cell lines, BBB permeability, and increased brain uptake in neuroinflammation mice qualifies [18F]FMTP as a potential PET tracer for studying inflammation.

Endometritis in the bitch: Immunohistochemical localization of cyclooxygenase 2.

Background:In several mammals, subfertility or infertility associated with endometritis was reported. Although there have been studies about endometritis in bitches, the pathophysiological mechanisms are not completely known.Aim:This study aimed to evaluate the immunohistochemical expression of Cyclooxygenase 2 (COX2) in clinically healthy bitches with normal uterine tissue and bitches with endometritis. Methods:Forty-eight mixed breed bitches in diestrus were used. Uterine biopsies were collected for diagnosis [normal endometrium (n = 15; NE), cystic endometrial hyperplasia (n = 1), atrophy (n= 2), acute endometritis (n = 9; AE), subacute endometritis (n = 7; SE), and chronic endometritis (n = 14; CE)]. Immunostaining and quantification of positively stained cells was performed on full-thickness uterine biopsies. Data were analyzed by the GLIMMIX procedure of SAS. Results:COX2 immunostaining was scattered and restricted to cells in the stroma in bitches with NE. However, in bitches with endometritis, strong staining was observed in the luminal epithelium, glandular epithelium, and stromal cells. Staining was also observed in inflammatory cells localized in the stroma as well as inside of the glands. The percentage of COX2 positive stromal cells in bitches with AE, SE, and CE was significantly higher compared with NE (p < 0.005). In addition, the percentage of COX2 positive stromal cells in bitches with SE, and CE was significantly lower compared with AE (p < 0.003). Conclusion:COX2 could be involved in the pathophysiological mechanisms producing endometritis without the presence of cystic endometrial hyperplasia in bitches. However, further researches on this topic are required.

Targeting Heterogeneous Tumors Using a Multifunctional Molecular Prodrug.

Reported here is a molecular construct (K1) designed to overcome hurdles associated with delivering active drugs to heterogeneous tumor environments. Construct K1 relies on two cancer environment triggers (GSH and H2O2) to induce prodrug activation. It releases an active drug form (SN-38) under conditions of both oxidative and reductive stress in vitro. Specific uptake of K1 in COX-2 positive aggressive colon cancer cells (SW620 and LoVo) was seen, along with enhanced anticancer activity compared with the control agent SN-38. These findings are attributed to environmentally triggered drug release, as well as simultaneous scavenging of species giving rise to intracellular redox stress. K1 serves to downregulate various cancer survival signaling pathways (AKT, p38, IL-6, VEGF, and TNF-α) and upregulate an anti-inflammatory response (IL-10). Compared with SN-38 and DMSO as controls, K1 also displayed an improved in vivo therapeutic efficacy in a xenograft tumor regrowth model with no noticeable systematic toxicity at the administrated dose. We believe that the strategy described here presents an attractive approach to addressing solid tumors characterized by intratumoral heterogeneity.

Combined Treatment of Cyclosporine and Mycophenolate Improves Endothelial Function in Aorta of LDLR−/− Mice

BackgroundChronic inflammation is a key player in the process of atherosclerosis. The present study was designed to investigate effects of immunosuppressive agents on endothelial dysfunction in a mouse model of atherosclerosis.MethodsTo induce atherosclerosis, 6‐week old LDLR knockout mice were fed with western diet for two months. Clinically used immunosuppressive agents, cyclosporine and mycophenolate, were mixed in chow. Aortic rings were fixed in the organ chambers for isometric tension measurement. Atherosclerotic plaques in aortic arch were visualized using Oil red O (ORO) staining and the level of CD68, COX‐2 positive cells was studied by Immunofluorescence. Lipoprotein and creatinine levels was measured using serum.Key resultsORO staining showed that atherosclerosis plaques in the aortic arch were reduced in mice fed with immunosuppressive drugs, compared with those fed with western diet. Acetylcholine induced relaxations at low concentrations, but contractions at high concentrations in the aorta of LDLR knockout mice. The acetylcholine‐induced contractions, but not the relaxations, were significantly reduced in mice treated with immunosuppressive agents. In the presence of indomethacin (non‐selective inhibitor of cyclooxygenase), acetylcholine‐induced contractions were restored to relaxations in both groups. The CD68, COX‐2 positive cells in the plaques of the two groups were not significantly different. In addition, there was no significant difference in serum lipoprotein and creatinine levels between the two groups of mice.ConclusionImmunosuppressive treatment reduces atherosclerotic plaque and prevents endothelial dysfunction in LDLR knockout mice. The underlying mechanisms may involve cyclooxygenase pathway; further studies are warranted to confirm, or not, the postulation.Support or Funding InformationThe present study was supported by the National Nature Science Foundation of China (81573418) and ZSYXRC‐014This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression and association of carbonic anhydrase IX and cyclooxygenase-2 in colorectal cancer

BackgroundThis work was designed to determine the relationship between hypoxia-inducible protein carbonic anhydrase IX (CA IX) and pro-inflammatory enzyme cyclooxygenase-2 (COX-2) in patients with colorectal cancer (CRC). MethodsWe examined CA IX and COX-2 expression in CRC tissues by immunohistochemical staining of 111 samples. We evaluated the correlation between the expression of these proteins and their correlation with the clinico-morphological parameters of CRC. ResultsCA IX was detected in 89 of 111 cases (80.2%). We predominantly observed membrane staining (70.3%) and a strong immunoreaction intensity (58.6%). The percentage of labeled cells in malignant lesions was less than 25% in 12.6% of cases, less than 50% in 15.3% of cases and more than 50% in 52.3% of CRC cases. The COX-2 protein was expressed in 94 of 111 cases (84.7%). We noticed only cytoplasmic localization, while immunoreaction intensity was predominantly strong (47.8%). The percentage of COX-2 positive cells was less than 25% only in 2.7% of the cases, less than 50% in 21.6% of the cases and more than 50% in 60.4% of the cases. No statistically significant correlations were observed between CA IX expression and clinico-morphological parameters. COX-2 expression was only significantly correlated with the tumor stage. Statistical analysis confirmed a significant correlation between the parameters of expression of the CA IX and COX-2 proteins. ConclusionCA IX/COX-2 interplay can promote hypoxia survival and the invasion of tumor cells. These proteins may represent independent prognostic factors of CRC.

Cyclooxygenase-2 expression in actinic keratosis.

IntroductionActinic keratosis (AK) is a common age-associated dermatosis typical for skin with photo-ageing features. Actinic keratosis lesions transform into invasive squamous cell carcinoma, if left untreated, but inductive factors remain unknown. A role of prostaglandins in the neoplastic process has been postulated.AimTo evaluate the possible correlation between cyclooxygenase-2 (COX-2) antigen expression in the immunohistochemical reaction and the stage of AK using the keratinocytic intraepidermal neoplasia (KIN) classification, age, sex and skin phenotype.Material and methodsSkin samples of AK were examined histopathologically using the KIN classification. Immunohistochemical analysis of COX-2 expression was conducted using a commercially available kit and Image Processing and Analysis in Java. The intensity was presented as the percentage of cells with a positive reaction.ResultsOut of the 94 subjects with AK (aged 51–93 years; mean: 76.51 ±9.64), 58 were female and 36 male. Phenotype 2 was found in 50 and phenotype 3 in 44 patients. Mean values of positive immunohistochemical reaction for COX-2 were 2.16 ±6.56% for women and 1.96 ±6.59% for men (2.47 ±6.61%), with no statistically significant difference. Mean values of anti-COX-2 antibody reaction were 2.30 ±6.82% in the KIN1 group and 2.48 ±7.01% in the KIN2 group, while no expression was found in all cases with KIN3. No statistically significant differences were found between the percentage of COX-2 positive cells in KIN1/KIN2.ConclusionsNo statistically significant correlations between the intensity of COX-2 reaction and AK stage were found. Cyclooxygenase-2 expression in AK is not affected by age, sex, or skin phenotype.

Quercetin attenuates the ischemia reperfusion induced COX-2 and MPO expression in the small intestine mucosa

Quercetin, the active substance of tea, fruits and vegetables, exerts a broad spectrum of pharmacological activities and is considered to have potential therapeutic application. The present study was designed to investigate the beneficial effect of quercetin against experimental ischemia- reperfusion (IR) injury of the small intestine in rats. Quercetin was administrated intraperitoneally 30min before 1h ischemia of superior mesenteric artery with following reperfusion periods lasting 1, 4 and 24h. The male specific pathogen-free Charles River Wistar rats were used (n=45). In acute phase, 4h after start of reperfusion, the quercetin induced a significant decrease in mucosal injury index (p<0.05) accompanied by a significant decrease in cyclooxygenase-2 (COX-2) expression in the epithelial lining of the intestinal villi in comparison with the control group (p<0.01). In the epithelium of the intestinal glands, COX-2 expression resulting from IR injury significantly increased regardless quercetin application (in control group p<0.001; in quercetin group p<0.05), but in quercetin group, significant decrease in it during 24h of reperfusion in a late phase of IR injury was detected (p<0.001). Based on morphology of COX-2 positive cells, the COX-2 positivity was found particularly in goblet cells of the intestinal villi epithelium and enteroendocrine cells respectively, in the glandular epithelium. We concluded that quercetin application attenuated mucosal damage from IR injury by inhibiting neutrophil infiltration which was demonstrated by a lower number of myeloperoxidase positive cells in the lamina propria during both phases of IR injury and the significant decrease in that in a late phase after 24h of reperfusion (p<0.05).

Uterine clinical findings, fertility rate, leucocyte migration, and COX-2 protein levels in the endometrial tissue of susceptible mares treated with platelet-rich plasma before and after AI

Persistent mating-induced endometritis (PMIE) results in decreased fertility in horses, thereby causing a significant impact in the horse market. Platelet-rich plasma (PRP), a modulator of the inflammatory response, has been largely used in veterinary medicine. Here, we investigated the effects of PRP on uterine inflammation, conception rate, endometrial polymorphonuclear neutrophil (PMN) migration, and COX-2 protein levels in the endometrial tissue. Thirteen PMIE-susceptible mares were used for artificial insemination (AI). The mares were inseminated with fresh semen in three consecutive cycles in a cross-over study design. The following cycle classifications were used: control cycle, no pharmacological interference; pre-AI, 20 mL of PRP was infused 24 h before AI; and post-AI, 20 mL of PRP was infused four h after AI. Follicular dynamics were monitored daily by transrectal ultrasound. When a follicle larger than 35 mm was detected, ovulation was induced with deslorelin acetate (1 mg, im). AI was performed 24 h after ovulation induction. Intrauterine fluid (FLU) was evaluated by ultrasonography before and 24 h after AI. PMNs in uterine cytology (CYT) and biopsy (HIS) were also observed before and 24 h after AI. Pregnancy was determined within 14 days after ovulation. Number of COX-2 positive cells was evaluated by immunohistochemistry. Both PRP treatments resulted in a decrease of PMNs in the CYT after breeding when compared to controls. FLU did not differ between cycles; however, the conception rates were significantly higher in the PRP mares. Mares positive for endometritis decreased in both treatment groups, and a more intense positive COX-2 labeling was observed in the control group when compared to the two treatment groups. In conclusion, PRP beneficially reduces inflammatory response in PMIE mares independent of when treatments were administered, thus increasing chances of successful pregnancy.

Radioiodinated indomethacin amide for molecular imaging of cyclooxygenase-2 expressing tumors.

Cyclooxygenase-2 (COX-2) is an important biomarker in several tumors. Available imaging probes display relatively low tumor to background ratios (smaller than 2:1). We evaluated newly developed indomethacin (Ind) derivatives for in vivo molecular imaging of COX-2 expressing carcinoma. Radioiodinated Ind derivatives Ind-NH-(CH2)4-NH-3-[I-125]I-Bz ([I-125]5), Ind-NH-(CH2)4-NH-5-[I-124/125]I-Nic ([I-124/125]6) and Ind-NH-(CH2)4-NH-5-[I-125]I-Iphth ([I-125]7) were prepared from the respective SnBu3-precursors (45–80% radiochemical yield; > 95% radiochemical purity). The cellular uptake of [I-125]5 and [I-125]6 correlated with COX-2 expression determined by SDS page/Western blot analysis. [I-125]5 was predominantly localized in the cell membrane while [I-125]6 was internalized and displayed a diffuse and favorable cytoplasmic distribution. In contrast, [I-125]7 showed only low uptake in COX-2 positive cells. Co-incubation with the COX-2 inhibitor Celecoxib led to an almost complete suppression of cellular uptake of [I-125]5 and [I-125]6. In vivo molecular imaging using positron emission tomography (PET) in SCID mice xenografted with COX-2+ (HT29) and COX-2− (HCT116) human colorectal carcinoma cells was performed for [I-124]6. HT29 xenografts displayed a significantly higher uptake than HCT-116 xenografts (5.6 ± 1.5 vs. 0.5 ± 0.1 kBq/g, P < 0.05) with an extraordinary high tumor to muscle ratio (50.3 ± 1.5). Immunohistological staining correlated with the imaging data. In conclusion, the novel radioiodinated indomethacin derivative ([I-124/125]6) could become a valuable tool for development of molecular imaging probes for visualization of COX-2 expressing tumors.

Cheongsangbangpung-tang ameliorated the acute inflammatory response via the inhibition of NF-\u03baB activation and MAPK phosphorylation

BackgroundCheongsangbangpung-tang (CBT) is a traditional herbal formula used in Eastern Asia to treat heat-related diseases and swellings in the skin. The present study was conducted to evaluate the anti-inflammatory effects of cheongsangbangpung-tang extract (CBTE) both in vitro and in vivo.MethodsThe in vitro effects of CBTE on the lipopolysaccharide (LPS)-induced production of inflammation-related proteins were examined in RAW 264.7 cells. The levels of nitric oxide (NO) were measured with the Griess reagent. Inflammatory cytokines and prostaglandin E2 (PGE2) were detected using the enzyme-linked immunosorbent assay (ELISA) method. Inflammation-related proteins were detected by Western blot. The effect of CBTE on acute inflammation in vivo was evaluated using carrageenan (CA)-induced paw oedema. To evaluate the anti-inflammatory effect, paw oedema volume, thickness of the dorsum and ventrum pedis skin, number of infiltrated inflammatory cells, and number of COX-2-, iNOS-immunoreactive cells were measured.ResultsIn an in vitro study, CBTE inhibited the production of NO and PGE2 and also decreased the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) activity, interleukin (IL)-1β, IL-6 and tumuor necrosis factor-α. In LPS-activated macrophages, nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) signalling is a pivotal pathway in the inflammatory process. These plausible molecular mechanisms increased the phosphorylation of I-κBα, while the activation of NF-κB and the phosphorylation of MAPK by LPS were blocked by CBTE treatment. In our in vivo study, a CA-induced acute oedematous paw inflammation rat model was used to evaluate the anti-inflammatory effect of CBTE. CBTE significantly reduced the increases in paw swelling, skin thicknesses, infiltrated inflammatory cells and iNOS-, COX-2 positive cells induced by CA injection.ConclusionsBased on these results, CBTE should favourably inhibit the acute inflammatory response through modulation of NF-κB activation and MAPK phosphorylation. Furthermore, the inhibition of CBTE in rat paw oedema induced by CA is considered to be clear evidence that CBTE may be a useful source to treat inflammation.