High Levels Of iNOS Research Articles

Relationship of serum inducible and endothelial nitric oxide synthase with exercise in healthy adult males and patients with type 2 diabetes mellitus.

The importance of physical activities in the control of diabetes mellitus disorder is well known but its correlation with endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in diabetic patients needs to be assessed. The aim of the study is to examine inducible and endothelial nitric oxide synthase in healthy adult male patients with type 2 diabetes mellitus (T2DM) in relation to exercise. This is a cross-sectional study that has been performed in the Department of Physiology, King Saud University, Riyadh, Saudi Arabia. Subjects were divided into Group 1: control (n=79), and Group 2: T2DM (n=85). Each group was subdivided into three subcategories: sedentary, moderate activity, and active subjects. Serum nitric oxide (NO), iNOS, eNOS, high sensitivity C-reactive protein (hs-CRP), nitrates, and nitrites, were compared between different groups. We observed significant differences in iNOS in diabetic patients compared to the control [29.1 (2.8) vs. 22.4 (1.4), p=0.050] with a significant decrease in eNOS when compared to the control group [79.5 (4.8) vs. 101.8 (5.7), p=0.003]. In the control group, eNOS showed significant differences (increase) between subgroups; sedentary, moderate, and active control subjects [77.4 (9.2), 114.7 (9.3), and 105.6 (9.5), p=0.026]. It also showed insignificant differences between subgroups in iNOS [23.4 (2.5), 24.2 (2.2), and 20.4 (2.4), p=0.520]. In addition, hsCRP showed a decrease with exercise but with no statistically significant difference between the groups [4.0 (0.5), 3.3 (0.3), and 2.9 (0.4), p=0.271]. In the diabetic patients' group, a significant difference (decrease) between subgroups in iNOS [43.5 (4.8), 20.8 (3.9), and 19.8 (4.6), p<0.001] and hsCRP [5.7 (0.5), 3.8 (0.4), and 3.6 (0.5), p=0.006] was detected. In addition, eNOS showed a decrease with exercise but with no statistically significant difference between groups [82.9 (7.8), 74.6 (7.9), and 81.7 (10.2), p=0.741]. Patients with T2DM have shown significantly low eNOS and high iNOS levels. Furthermore, regular exercise can significantly decrease iNOS in patients with T2DM. Therefore, the importance of exercise for diabetic patients has to be emphasized to prevent long-term complications related to diabetes mellitus.

Infection and innate immune mechanism of goose astrovirus.

Goose astrovirus (GAstV, genus Avian Astrovirus, family Astrovirus) was first discovered in 2005, but was not considered as a pathogen of gosling gout until 2016. Since then, goose astrovirus has erupted in Chinese goslings, causing at most 50% of gosling deaths. By December 2022, the disease had become epidemic and prevailed in goose farms in Jiangsu, Shandong, Anhui, Henan, Guangdong, Liaoning, Sichuan and other places in China. The disease mainly affects goslings within 3 weeks old. The typical symptoms of goose astrovirus are large deposits of urate in the viscera, joint cavity and ureter surface of infected goslings. Goose astrovirus infection can trigger high levels of iNOS, limiting goose astrovirus replication. The ORF2 domain P2 of the goose astrovirus activates the OASL protein, limiting its replication. Goose astrovirus can also activate pattern recognition receptors (RIG-I, MDA-5, TLR-3), causing an increase in MHC-Ia, MHC-Ib and CD81 mRNA, activating humoral and cellular immunity, thereby hindering virus invasion. Goose astrovirus also regulates the activation of IFNs and other antiviral proteins (Mx1, IFITM3, and PKR) in the spleens and kidneys to inhibit viral replication. The innate immune response process in goslings also activates TGF-β, which may be closely related to the immune escape of goose astrovirus. Gaining insight into the infection and innate immune mechanism of goose astrovirus can help researchers study and prevent the severe disease in goslings better.

Perivascular Macrophages Regulate Blood Flow Following Tissue Damage.

[Figure: see text].

Sildenafil Alleviates Murine Experimental Autoimmune Encephalomyelitis by Triggering Autophagy in the Spinal Cord.

Multiple Sclerosis (MS) is a neuroinflammatory and chronic Central Nervous System (CNS) disease that affects millions of people worldwide. The search for more promising drugs for the treatment of MS has led to studies on Sildenafil, a phosphodiesterase type 5 Inhibitor (PDE5I) that has been shown to possess neuroprotective effects in the Experimental Autoimmune Encephalomyelitis (EAE), an animal model of MS. We have previously shown that Sildenafil improves the clinical score of EAE mice via modulation of apoptotic pathways, but other signaling pathways were not previously covered. Therefore, the aim of the present study was to further investigate the effects of Sildenafil treatment on autophagy and nitrosative stress signaling pathways in EAE. 24 female C57BL/6 mice were divided into the following groups: (A) Control - received only water; (B) EAE - EAE untreated mice; (C) SILD - EAE mice treated with 25mg/kg of Sildenafil s.c. The results showed that EAE mice presented a pro-nitrosative profile characterized by high tissue nitrite levels, lowered levels of p-eNOS and high levels of iNOS. Furthermore, decreased levels of LC3, beclin-1 and ATG5, suggests impaired autophagy, and decreased levels of AMPK in the spinal cord were also detected in EAE mice. Surprisingly, treatment with Sildenafil inhibited nitrosative stress and augmented the levels of LC3, beclin-1, ATG5, p-CREB and BDNF and decreased mTOR levels, as well as augmented p-AMPK. In conclusion, we propose that Sildenafil alleviates EAE by activating autophagy via the eNOS-NO-AMPK-mTOR-LC3-beclin1-ATG5 and eNOS-NO-AMPK-mTOR-CREB-BDNF pathways in the spinal cord.

Macrophage phenotypes and monocyte subsets after destabilization of the medial meniscus in mice.

Macrophages play an important role in the development and progression of osteoarthritis (OA). The aim of this study was to identify macrophage phenotypes in synovium and monocyte subsets in peripheral blood in C57BL/6 mice by destabilizing the medial meniscus (DMM), and the association of macrophage subsets with OA features. DMM, sham, and non‐operated knees were histologically assessed between 1 and 56 days for macrophage polarization states by immunohistochemistry (IHC), cartilage damage, synovial thickening, and osteophytes (n = 9 per timepoint). Naive knees (n = 6) were used as controls. Monocyte and polarized synovial macrophage subsets were evaluated by flow cytometry. CD64 and CD206 levels on IHC were higher at early timepoints in DMM and sham knees compared to naive knees. iNOS labeling intensity was higher in DMM and sham knees than in naive knees from d3 onwards. CD163 expression was unaltered at all timepoints. Even though macrophage polarization profiles were similar in DMM and sham knees, only in DMM knees the presence of iNOS and CD206 associated with synovial thickness, and CD163 staining inversely correlated with osteophyte presence. At day 14, monocyte subset distribution was different in peripheral blood of DMM mice compared with sham mice. In conclusion, monocyte subsets in blood and synovial macrophage phenotypes vary after joint surgery. High levels of iNOS+, CD163+, and CD206+ cells are found in both destabilized and sham‐operated knees, and coexistence with joint instability may be a requirement to initiate and exacerbate OA progression.

Inhibition of mitochondrial fission and iNOS in the dorsal vagal complex protects from overeating and weight gain

ObjectivesThe dorsal vagal complex (DVC) senses insulin and controls glucose homeostasis, feeding behaviour and body weight. Three-days of high-fat diet (HFD) in rats are sufficient to induce insulin resistance in the DVC and impair its ability to regulate feeding behaviour. HFD-feeding is associated with increased dynamin-related protein 1 (Drp1)-dependent mitochondrial fission in the DVC. We investigated the effects that altered Drp1 activity in the DVC has on feeding behaviour. Additionally, we aimed to uncover the molecular events and the neuronal cell populations associated with DVC insulin sensing and resistance. MethodsEight-week-old male Sprague Dawley rats received DVC stereotactic surgery for brain infusion to facilitate the localised administration of insulin or viruses to express mutated forms of Drp1 or to knockdown inducible nitric oxide synthase (iNOS) in the NTS of the DVC. High-Fat diet feeding was used to cause insulin resistance and obesity. ResultsWe showed that Drp1 activation in the DVC increases weight gain in rats and Drp1 inhibition in HFD-fed rats reduced food intake, weight gain and adipose tissue. Rats expressing active Drp1 in the DVC had higher levels of iNOS and knockdown of DVC iNOS in HFD-fed rats led to a reduction of food intake, weight gain and adipose tissue. Finally, inhibiting mitochondrial fission in DVC astrocytes was sufficient to protect rats from HFD-dependent insulin resistance, hyperphagia, weight gain and fat deposition. ConclusionWe uncovered new molecular and cellular targets for brain regulation of whole-body metabolism, which could inform new strategies to combat obesity and diabetes.

Role of nitric oxide in the response to photooxidative stress in prostate cancer cells

A continuous state of oxidative stress during inflammation contributes to the development of 25% of human cancers. Epithelial and inflammatory cells release reactive oxygen species (ROS) and reactive nitrogen species (RNS) that can damage DNA. ROS/RNS have biological implications in both chemoresistance and tumor recurrence. As several clinically employed anticancer drugs can generate ROS/RNS, we have addressed herein how inducible nitric oxide synthase and nitric oxide (iNOS/•NO) affect the molecular pathways implicated in the tumor response to oxidative stress. To mimic the oxidative stress associated with chemotherapy, we used a photosensitizer (pheophorbide a) that can generate ROS/RNS in a controlled manner. We investigated how iNOS/•NO modulates the tumor response to oxidative stress by involving the NF-κB and Nrf2 molecular pathways. We found that low levels of iNOS induce the development of a more aggressive tumor population, leading to survival, recurrence and resistance. By contrast, high levels of iNOS/•NO sensitize tumor cells to oxidative treatment, causing cell growth arrest. Our analysis showed that NF-κB and Nrf2, which are activated in response to oxidative stress, communicate with each other through RKIP. For this critical role, RKIP could be an interesting target for anticancer drugs. Our study provides insight into the complex signaling response of cancer cells to oxidative treatments as well as new possibilities for the rational design of new therapeutic strategies.

Abstract P3-03-05: Elucidating the synergistic relationship between the iNOS and PI3K/Akt pathways for treatment of metaplastic breast cancer

Abstract Metaplastic breast cancer (MBC) is a rare form of breast cancer characterized by histological presence of two or more cellular types, typically epithelial and mesenchymal components. MBC represents 0.2 to 5% of all breast cancers. Most MBC typically displays a triple-negative breast cancer phenotype in which they lack expression of ER, PR, and HER2 and they also exhibit basal-like or claudin-like molecular subtypes. The current treatment guidelines for MBC have yet to be established due to limited knowledge of its pathogenesis. Prior studies have shown that MBC harbor a subtype mutation in RPL39, which is correlated with higher levels of iNOS, and aberrations in the PI3K/Akt signaling pathway. Considering that aberrations in both of these pathways may play a role in disease pathogenesis, our goal is to further elucidate whether there is a synergistic relationship between the iNOS and PI3K/Akt pathways in MBC. Our hypothesis is that isoform-specific PIK3CA inhibitor Alpelisib, will interact synergistically with pan-NOS inhibitor L-NMMA, to act as an improved therapeutic combination against MBC. We used MBC cell lines Hs578t and BT549 and MBC PDX models RPL39-positive BCM 4664 and RPL39-negative BCM 3807 in our preliminary studies. Using flow cytometry to detect Annexin V+/DAPI+ cells, we found increased levels of cell death in MBC cell lines treated with LNMMA+Alpelisib combination treatment in comparison to single agent treatment. Western blot analysis of samples from single (LNMMA or Alpelisib) or combination treated cell lines and PDXs showed increased PARP degradation and cleaved caspase 3/9 with combination treatment. Pathway-focused RT-PCR analysis was performed using RNA collected from Hs578t cells treated for 48 hrs. with LNMMA, Alpelisib, or combination treatment. RT-PCR data were analyzed using Ingenuity Pathway Analysis (IPA; Qiagen) software which showed that insulin signaling and PI3K/AKT pathways were the most enriched pathways with single and combination treatment. In vivo data using PDX BCM 4664 showed that combination treatment of LNMMA+Alpelisib was most effective at reducing tumor volume in comparison to single treatment. Our preliminary results suggest that LNMMA and Alpelisib combination treatment have therapeutic potential in the treatment of MBC. Our results indicated that simultaneous inhibition of the NOS and PI3K/AKT pathways may represent a clinically relevant therapeutic concept against hard-to-treat MBC, and warrants further studies. Citation Format: Tejaswini Parlapalle Reddy, Roberto Rosato, Liliana Guzman, Wei Qian, Jianying Zhou, Anthony J Kozielski, Jenny C Chang. Elucidating the synergistic relationship between the iNOS and PI3K/Akt pathways for treatment of metaplastic breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-05.

Hepatic cytochrome P450 metabolism suppressed by mast cells in type 1 allergic mice

Mast cells and Kupffer cells secrete interleukin (IL)-1β, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, which stimulate excess nitric oxide (NO) producing-inducible NO synthase (iNOS). Unlike Kupffer cells, immunoglobulin E-sensitized mast cells elicit sustained NO production. We investigated the participation of mast cell-released NO and cytokine-derived iNOS activation in type 1 allergy-suppressed hepatic cytochrome P450 (CYP) metabolism. Aminoguanidine, a selective iNOS inhibitor, completely suppressed serum nitrate plus nitrite (NOx) concentrations after primary and secondary sensitization of ICR mice and markedly attenuated allergy-suppressed hepatic CYP1A2, CYP2C, CYP2E1, and CYP3A activities. In the liver, primary and secondary sensitization enhanced iNOS-stimulating IFN-γ (5-15-fold) and TNF-α (3-5-fold) mRNA levels more than IL-1β (2-fold) and F4/80-positive Kupffer cell (2-fold) mRNA levels. When mast cell-deficient (−/−) mice were sensitized, hepatic CYP activities were not suppressed. Serum NOx levels in the sensitized −/− mice were similar with those in saline-treated ICR and −/− mice. In the liver of −/− mice, secondary sensitization markedly enhanced mRNA expression of iNOS (20-fold), IFN-γ (15-fold), and TNF-α (3-fold). However, hepatic total NOS activities in −/− mice were not significantly different between saline treatment and sensitization. Similarly, primary and secondary ICR mice did not significantly enhance total NOS activities in the liver and hepatocytes. The total NOS activities observed did not relate to the high levels of iNOS, IFN-γ, and TNF-α mRNA in the liver. Hepatic c-kit-positive mast cells in sensitized ICR mice were maintained at control levels. Therefore, our data suggest that mast cell-released NO participates in type 1 allergy-suppressed CYP1A2, CYP2C, CYP2E1, and CYP3A metabolism.

Abstract A128: Imprime PGG, a novel, clinical-stage pathogen associated molecular pattern, modulates MDSC function, facilitating a coordinated antitumor immune response

Abstract The success of cancer immunotherapy is limited by multiple mechanisms of tumor-induced immunosuppression often a result of suppressive myeloid cells. M2-like tumor associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), and tolerogenic dendritic cells (DC) constitute this immunosuppressive tumor microenvironment (TME) and have repeatedly been associated with poorer prognoses. Therapies designed to overturn this suppressive immune microenvironment could substantially enhance the efficacy of multiple immunotherapeutic approaches. Imprime PGG (Imprime) is a yeast β-1,3/1,6 glucan that has shown compelling efficacy in multiple Phase II trials with tumor-targeting and anti-angiogenic antibodies. As a pathogen associated molecular pattern (PAMP), Imprime activates myeloid cells (monocytes/macrophages, neutrophils, dendritic cells). Our previous ex vivo human and in vivo mouse studies have shown that Imprime promotes repolarization of M2 macrophages to an anti-tumor, M1-like orientation and enhances DC maturation, driving T cell expansion and the production of interferon gamma (IFNγ). We now show that Imprime can modulate the function of immature myeloid cells- the myeloid derived suppressor cells. MDSCs were generated from human cord blood by purifying CD34+ cells and culturing with GMCSF or GCSF ± tumor conditioned media. After 9 days, HLA-DR cells were depleted. The remaining cells were isolated and treated with Imprime or vehicle control. We found that incubating tumor-conditioned, vehicle-treated MDSCs with CD3/CD28-activated CD8 or CD4 T cells inhibited T cell proliferation. Imprime treatment significantly reduced this inhibition. Phenotypically, Imprime treatment elicited substantially increased expression of the co-stimulatory molecules CD80 and CD86 in both monocytic (Mo) and granulocytic (PMN) MDSCs, suggesting that these MDSCs now exhibited APC-like characteristics. In in vivo murine tumor models, Imprime was also able to modulate MDSC phenotype and function, which coincided with enhanced anti-tumor efficacy. Specifically, in the H441 human NSCLC xenograft model, Imprime when combined with 10mg/kg DC101 (murine anti-VEGFR2 Ab), significantly repressed tumor growth compared to the DC-101 alone or vehicle groups. Moreover, spleens from Imprime + DC101 treated-mice had reduced numbers of immunosuppressive, splenic Mo- or PMN-MDSC compared to either DC101 alone or vehicle control. The splenic Mo- or PMN-MDSCs from Imprime + DC101 treated mice that were present expressed significantly higher CD86. The Mo-MDSCs also expressed higher levels of iNOS, a key marker for activated macrophages. In another xenograft NSCLC model, H1299, the splenic MDSC after treatment with Imprime and α-VEGF-A Ab, bevacizumab (bev), showed increased iNOS expression and reduced Arg-1 expression- a shift typifying the M1 polarization state. Collectively, these data show that Imprime treatment can reshape the suppressive immune microenvironment of the tumor and elicit a robust anti-tumor immunity by targeting multiple myeloid lineage cells. Citation Format: Kathryn Fraser, Anissa Chan, Xiohong Qiu, Nadine Ottoson, Adria Bykowski Jonas, Jeremy Graff, Nandita Bose. Imprime PGG, a novel, clinical-stage pathogen associated molecular pattern, modulates MDSC function, facilitating a coordinated antitumor immune response [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A128.

Novel mechanism in autoimmune regulation by S-nitrosoglutathione

Abstract Nitric oxide (NO) plays a pivotal role in pathogenesis and control of autoimmune disease which is caused by abnormal activation of T helper (Th) cells. Under disease conditions of experimental autoimmune encephalomyelitis (EAE), we observed that different subsets of Th cells expressed different levels of NO synthesizing enzymes (nNOS, iNOS, and eNOS). Notably, Th17 cells exclusively expressed high levels of iNOS and its genetic ablation resulted in increased Th17 activation and IL-17 expression but decreased activation of specific subset of regulatory T (CD4+CD25+FOXP− Treg) cells and IL-10 expression in EAE. S-nitrosoglutathione (GSNO) is a physiological NO carrier molecules that mediates variety of NO signaling via trans-S-nitrosylation mechanisms. In EAE, GSNO treatment decreased the disease severity and activation/effector function of Th17 while it increased CD4+CD25+FOXP− Treg activation/effector function. Moreover, the reduced activation/effector function of Th17 by adaptive transfer of GSNO-induced CD4+CD25+FOXP− Tregs suggest that CD4+CD25+FOXP− Treg may be the effector cells antagonizing Th17 in response to iNOS/NO/GSNO. The cellular levels of GSNO is regulated by its catabolic enzyme GSNO reductase (GSNOR). In EAE, both Th17 and of CD4+CD25+FOXP− Treg expressed high levels of GSNOR and pharmacological inhibition of GSNOR or genetic ablation of GSNOR resulted in abrogation of Th17 activation/effector function while activating CD4+CD25+FOXP− Treg. In summary, this study suggest the iNOS/NO/GSNO-inducible CD4+CD25+FOXP− Treg cells as the potential feedback regulator of Th17 and GSNOR as the potential therapeutic target for Th17 and CD4+CD25+FOXP− Treg mediated autoimmune disease.

Activation of mesenchymal stem cells by macrophages promotes tumor progression through immune suppressive effects.

Cancer development and progression is linked to tumor-associated macrophages (TAMs). Distinct TAMs subsets perform either protective or pathogenic effects in cancer. A protective role in carcinogenesis has been described for M1 macrophages, which activate antitumor mechanisms. By comparison, TAMs isolated from solid and metastatic tumors have a suppressive M2-like phenotype, which could support multiple aspects of tumor progression. Currently, it has not been clearly understood how macrophages in tumor-associated stroma could be hijacked to support tumor growth. Mesenchymal stem cells (MSCs) actively interact with components of the innate immune system and display both anti-inflammatory and pro-inflammatory effects. Here, we tested whether MSCs could favor the tumor to escape from immunologic surveillance in the presence of M1 macrophages. We found that MSCs educated by M1 condition medium (cMSCs) possessed a greatly enhanced ability in promoting tumor growth in vivo. Examination of cytokines/chemokines showed that the cMSCs acquired a regulatory profile, which expressed high levels of iNOS and MCP1. Consistent with an elevated MCP1 expression in cMSCs, the tumor-promoting effect of the cMSCs depended on MCP1 mediated macrophage recruitment to tumor sites. Furthermore, IL-6 secreted by the cMSCs could polarize infiltrated TAMs into M2-like macrophages. Therefore, when macrophages changed into M1 pro-inflammation type in tumor microenvironment, the MSCs would act as poor sensors and switchers to accelerate tumor growth.

Radiation enhances the therapeutic effect of Banoxantrone in hypoxic tumour cells with elevated levels of nitric oxide synthase.

Banoxantrone (AQ4N) is a prototype hypoxia selective cytotoxin that is activated by haem containing reductases such as inducible nitric oxide synthase (iNOS). In the present study, we evaluate whether elevated levels of iNOS in human tumour cells will improve their sensitivity to AQ4N. Further, we examine the potential of radiation to increase cellular toxicity of AQ4N under normoxic (aerobic) and hypoxic conditions. We employed an expression vector containing the cDNA for human iNOS to transfect human fibrosarcoma HT1080 tumour cells. Alternatively, parental cells were exposed to a cytokine cocktail to induce iNOS gene expression and enzymatic activity. The cells were then treated with AQ4N alone and in combination with radiation in the presence or absence of the iNOS inhibitor N-methyl-L-arginine. In parental cells, AQ4N showed little difference in toxicity under hypoxic verses normoxic conditions. Notably, cells with upregulated iNOS activity showed a significant increase in sensitivity to AQ4N, but only under conditions of reduced oxygenation. When these cells were exposed to the combination of AQ4N and radiation, there was much greater cell killing than that observed with either modality alone. In the clinical development of hypoxia selective cytotoxins it is likely they will be used in combination with radiotherapy. In the present study, we demonstrated that AQ4N can selectively kill hypoxic cells via an iNOS-dependent mechanism. This hypoxia-selective effect can be augmented by combining AQ4N with radiation without increasing cytotoxicity to well-oxygenated tissues. Collectively, these results suggest that targeting hypoxic tumours with high levels of iNOS with a combination of AQ4N and radiotherapy could be a useful clinical therapeutic strategy.

Abstract 18939: Micro RNA-93 Promotes Macrophage M2 Polarization in Experimenal Peripheral Arterial Disease

Macrophages (MØ) were well known to play vital roles in promoting blood flow by arteriogenic induction in experimental Peripheral Arterial Disease (PAD). MØs depending on the cytokine milieu, exist in M1 or M2 polarization states (two extremes of polarization continuum). While M1-MØs are pro-inflammatory and anti-angiogenic, M2-MØs are anti-inflammatory and pro-angiogenic. We have previously demonstrated that Micro RNA-93 (Mir93) promotes angiogenesis and perfusion recovery in experimental PAD. However, the downstream mechanisms that operate in Mir93 mediated perfusion recovery in ischemic leg are not yet clear. In our current study we investigated the role of Mir93 in MØ polarization in vitro and in animal models of hind limb ischemic (HLI). We hypothesized that “Mir93 induction in ischemic muscle promotes M2-MØ polarization that improves tissue repair, survival and neovascularization in PAD” Mir93 mimic transfection in Raw264.7 (MØ cell line) significantly induced Arginase-1 (M1 marker) expression and decreased inducible Nitric oxide synthase (iNOS) (M2 marker) expression compared to negative mimic in qPCR analysis. Bone marrow derived macrophages (BMDM) from MCM7-KO (Mir93 deficient mice) express significantly higher levels of iNOS compared to littermates under hypoxia serum starvation. Monocytes isolated from the bone marrow of MCM7-KO ischemic legs express significantly higher levels of iNOS compared to littermates. Conditioned medium from Antagomir-93 treated Raw264.7 MØs and MCM7-KO BMDM significantly decreased HUVEC capillary like tube formation on matrigel compared to negative antagomir treated or littermates. Flow cytometry analysis showed that MCM7-KO mice had significantly higher levels of M1-MØs correlating with decreased capillary density compared to litter mate controls. P&lt;0.05 considered significant We conclude that Mir93 plays a crucial role in inducing MØ-M2 polarization. Increased Mir93 levels in ischemic hind limb promote M2-MØ polarization that helps in recovery of ischemic leg by tissue repair and neovascularization. Ongoing experiments are aimed to determine whether Mir93 deficient MØs can inhibit perfusion recovery in littermates and the downstream targets of Mir93 that regulate MØ polarization in HLI

The role of nitric oxide and oxidative stress in intestinal damage induced by selenium deficiency in chickens.

Nitric oxide (NO) is an essential messenger molecule and is associated with inflammation and oxidative stress. Although NO has important biological functions in mammals, its role in the mechanism that occurs after intestinal injuries in chickens remains unknown. The objective of the present study was to investigate the real role of NO and oxidative stress in the intestinal injuries of chickens induced by selenium (Se) deficiency. A total 150 chickens were randomly divided into the following two groups: a low-Se group (L group, fed a Se-deficient diet containing 0.020mg/kg Se) and a control group (C group, fed a commercial diet containing 0.2mg/kg Se). The activities and mRNA levels of glutathione peroxidase (GSH-Px), the production of glutathione (GSH) and NO, and the protein and mRNA levels of inducible nitric oxide synthase (iNOS) were examined in the intestinal tissues (duodenum, jejunum, and rectum) at 15, 25, 35, 45, and 55days. Methane dicarboxylic aldehyde (MDA) levels were also detected by assay kits. Then, the morphologies of the tissues were observed under the microscope after hematoxylin and eosin staining (H&E staining). The results showed that Se deficiency induced higher inflammatory damage and MDA levels (P < 0.05), which were accompanied by higher levels of iNOS and NO but lower levels of GSH and GSH-Px (P < 0.05). Our results indicated that Se deficiency induced oxidative damage in the intestinal tracts of chickens and that low levels of GSH-Px and high contents of NO may exert a major role in the injury of the intestinal tract induced by Se deficiency.

INOS and eNOS Genotypes and Expression in Hepatitis E-Related Acute Liver Failure

Introduction: The study was aimed to determine the role of iNOS and eNOS genotypes in their expression in hepatitis E virus (HEV)-related acute liver failure (ALF). Methods: The study group included 294 acute viral hepatitis (AVH), 82 ALF and 331 healthy controls. Expression study of iNOS and eNOS was done using commercially available ELISA kits. Polymorphism of iNOS C150T and eNOS G894T was studied using the PCR-RFLP method. Determination of the iNOS and eNOS gene mutations was done using the TSP509I restriction enzyme for iNOS and Ban II restriction enzyme for eNOS. Pearson correlation analysis was done to observe the correlations between iNOS and eNOS with the parameters for the severity of disease (viral load, prothrombin time, total bilirubin, SGOT, SGPT, and ALP). Results: On comparing the iNOS levels corresponding to its genotype in the study group, we observed that the subjects carrying the CT+TT mutant genotype had significantly higher levels of iNOS compared to the wild type CC genotype (24.9± 6.9 vs.16.74±5.1; p<0.001, 62.16±1.6 vs. 54.26 ±6.1, p<0.001, and 10.57±5.15 vs.7.36±2.5, p<0.001). Similarly, on comparing the eNOS levels corresponding to its genotype in the study groups, we observed that the subjects carrying the GT+TT mutant genotype had significantly higher levels of eNOS compared to the wild type GG genotype (27.3± 7.2 vs.21.45±5.1, p<0.001, 64.1±1.8 vs. 58.5 ±2.8, p<0.001 and 17.9±4.8 vs.14.6±4.3, p<0.001). Significant positive correlation was observed between the iNOS and eNOS levels in the HEV-infected patient (AVH and ALF) and the severity of disease parameters i.e viral load, prothrombin time, total bilirubin, direct bilirubin, SGOT, and SGPT levels. Conclusion: iNOS and eNOS mutant genotype significantly increases the levels of iNOS and eNOS, as compared to wild type. The iNOS C150T polymorphism and the eNOS G894T polymorphism and high levels of iNOS and eNOS are associated with an increased risk of HEV-related acute hepatitis and liver failure.

Abstract 3664: High frequency of a circulating monocytic subpopulation of myeloid-derived suppressor cells predicts worst clinical outcome in untreated non-small lung cancer patients

Abstract Background: The immune system can either prevent and control the tumor growth or promote tumor escape. Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of immature cells with immune suppressive properties and their overexpression seems to confer a worse prognosis in cancer patients. Objective: The aim of the study was to investigate the presence of new phenotypically immature subpopulations of MDSCs which could be associated with advanced/metastatic NSCLC patients' clinical outcome. Materials &amp; Methods: Circulating subtypes of MDSCs (monocytic and granulocytic), as well as the CD4+ , CD8+ , dendritic (DC), monocytes and B cells were assessed in peripheral blood of of chemotherapy-naive patients with advanced/metastatic NSCLC (n=134) and healthy controls (n=28) were analyzed using flow cytometry. The phenotypic characterization of MDSCs subpopulations was determined in strictly immature myeloid cells. A correlation between the levels of MDSCs and other tested immune cells as well as their association with overall (OS) and progression- free survival (PFS) was evaluated; high expression of MDSCs was defined as the percentage of the cells above the 90% percentile of the controls. Results: Two monocytic [M-MDSC: CD14+CD15-CD11b+CD33+HLA-DR-Lin- and CD14+CD15+CD11b+CD33+HLA-DR-Lin-] and a granulocytic [G-MDSC: CD14-CD15+CD11b+CD33+HLA-DR-Lin-] subpopulation of MDSCs were significantly increased (p&amp;lt;0.001), while the frequencies of CD4+ cells, DC and monocytes were decreased in patients compared to healthy donors (p≤0.0001, p≤0.0004, p=0.015; respectively). The frequency of both M-MDSCs' subpopulations was inversely correlated to that of DCs (p≤0.04), while the G-MDSC subpopulation was inversely correlated to CD4+ T cells (p=0.006). Furthermore, the M-MDSCs expressed high levels of iNOS, whereas the G-MDSCs generated high levels of reactive oxygen species (ROS). Increased frequencies of M-MDSCs were associated with worse response to front-line treatment (p=0.02) and patients with normal levels of CD14+CD15+CD11b+CD33+HLA-DR-Lin- MDSCs had longer OS and PFS compared to those with high levels (p=0.008 and p=0.02, respectively). Multivariate analysis revealed that the increased number of CD14+CD15+CD11b+CD33+HLA-DR-Lin- MDSCs was independently associated with decreased PFS and OS. Conclusion: The data provide evidence that the increased frequency of two new M-MDSC subpopulations in patients with advanced/metastatic NSCLC is associated with an unfavorable clinical outcome. Citation Format: Eleni Kyriaki Vetsika, Filippos Koinis, Marianthi Gioulmpasani, Despoina Aggouraki, Anna Koutoulaki, Eirini Skalidaki, Dimitris Mavroudis, Vassilis Georgoulias, Athanasios Kotsakis. High frequency of a circulating monocytic subpopulation of myeloid-derived suppressor cells predicts worst clinical outcome in untreated non-small lung cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3664. doi:10.1158/1538-7445.AM2014-3664

Role of nitric oxide synthase genes in hepatitis E virus infection

Hepatitis E virus (HEV) is the most common cause of endemic and epidemic acute hepatitis. A correlation between iNOS, eNOS polymorphisms, levels and severity of disease has been reported, and here, we examined the role of iNOS and eNOS gene polymorphisms and their levels in HEV-related acute viral hepatitis and acute liver failure. Hepatitis E virus-related cases of acute hepatitis (294 patients) and liver failure (82 patients) and age- and sex-matched healthy controls (331 subjects) were included in the study. PCR-RFLP was performed to identify the polymorphisms in the iNOS and eNOS genes. iNOS and eNOS levels were studied using ELISA assays and HEV viral load, genotype and combined effects of iNOS genotype, levels and parameters for disease severity were examined. The frequency of iNOS (CT + TT) and eNOS (GT + TT) genotypes was higher in subjects with liver failure compared with controls. iNOS and eNOS levels in patients with acute liver failure (55.51 ± 6.33 IU/mL, 60.2 ± 3.69) cases were significantly increased as compared to patients with acute viral hepatitis (17.8 ± 6.08 IU/mL, 23.7 ± 6.57) and controls (P < 0.05). A significant positive correlation was observed between the iNOS and eNOS levels in our study population when compared with the severity of disease parameters. Hence, the iNOS C150T polymorphism and the eNOS G894T polymorphism and high levels of iNOS and eNOS are associated with an increased risk of HEV-related acute hepatitis and liver failure. This study supports the possible role of nitric oxide synthase genes (iNOS and eNOS) in determining the severity of HEV infection.

Lipopolysaccharide-Induced M2 to M1 Macrophage Transformation for IL-12p70 Production Is Blocked by Candida albicans Mediated Up-Regulation of EBI3 Expression

Macrophages are heterogeneous cell populations that are present in all tissues. Macrophages can be divided into classically activated inflammatory macrophages (M1) and alternatively activated anti-inflammatory macrophages (M2). It has been generally accepted that M1 macrophages are polarised in an inflammatory environment to produce pro-inflammatory cytokines, whilst M2 macrophages are involved in anti-inflammation and aid tissue repair in wound healing. Bacterial endotoxin (lipopolysaccharide; LPS) is a potent factor in infection, which induces M1 macrophages resulting in higher levels of iNOS, TNFα and IL-12p70 which dictate inflammatory T cell responses. M2 macrophages can be transformed into M1 macrophages following LPS stimulation to promote inflammation. Candida albicans is a commensal fungal microorganism, which has been suggested to induce immune tolerance; however, the mechanism of C. albicans-induced immune tolerance has not been investigated in detail. IL-35 is a recently identified anti-inflammatory cytokine which is a heterodimeric protein consisting of the Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 shares the protein subunit p35, with IL-12p70. IL-12p70 is the most potent cytokine to induce Th1 responses during inflammation. In this study, we demonstrate that heat-killed C. albicans (HKC) strongly suppressed LPS-induced IL-12p70 production in M2 macrophages. Candida albicans induced a high level of EBI3 expression in M2 macrophages, which served as a mechanism for IL-12p70 suppression by competitive binding of the common protein subunit (p35) of IL-35 and IL-12p70. To demonstrate that EBI3 expression had the ability to block IL-12p70 production intracellularly, a Chinese Hamster Ovary (CHO) cell line with biscistronic expression of IL-12p40 and p35 was constructed, followed by ectopic over-expression of EBI3. The over-expression of EBI3 in the IL-12p70 producing cell line effectively suppressed IL-12p70 production. IL-35 secretion was also detected in the cell line, with suppressed IL-12p70 production by immune-precipitation Western blotting. However, this secretion was not evident in M2 macrophages following stimulation by HKC. This can be explained by the constitutive expression of IL-35 receptors (gp130 and IL-12Rβ2) in M2 macrophages for cytokine consumption. Our results have indicated that C. albicans can suppress host inflammatory responses in mucosal skin by suppressing LPS-induced IL-12p70 production. Lower IL-12p70 production may avoid an unnecessary Th1 response in order to retain immune tolerance, which may be one of the mechanisms by which C. albicans achieves a successful commensal lifestyle without having a detrimental effect on the host’s health.

Sodium L‐lactate differently affects brain‐derived neurothrophic factor, inducible nitric oxide synthase, and heat shock protein 70 kDa production in human astrocytes and SH‐SY5Y cultures

The present study analyzed the in vitro effects induced by sodium L-lactate on human astrocytes and the SH-SY5Y cell line, when added at concentrations of 5, 10, and 25 mmol/liter. Expression of brain-derived neurotrophic factor (BDNF), inducible nitric oxide synthase (iNOS), and heat shock protein 70 kDa (HSP70) was evaluated by Western blot analysis. Cell viability with MTT, release of nitric oxide (NO) through the Griess reaction, and production of BDNF by enzyme-linked immunoassay was determined. Data indicate that, in SH-SY5Y as well as in cortical astrocytes, after 4 hr sodium L-lactate increases the expression and release of BDNF, iNOS, and NO; after 24 hr, it turns is ineffective for the production of the neurotrophin in SH-SY5Y and not in astrocytes, but the expression of iNOS and release of NO appear to be further increased compared with those after 4 hr. Sodium L-lactate influences differently the expression of HSP70 in SH-SY5Y compared with astrocytes. We propose, based on these findings, that sodium L-lactate affects the expression of BDNF in SH-SY5Y and astrocytes in a different manner: high levels of iNOS and NO expressed in SH-SY5Y have a profound inhibitory effect on the release of BDNF related to a more limited production of HSP70 by SH-SY5Y. In conclusion, the results demonstrate differences in the responses of SH-SY5Y and astrocytes to stimulation by high levels of sodium L-lactate. Sodium L-lactate differently and dose and time dependently influences the expression and release of BDNF, iNOS, NO, and HSP70 depending on the cell type.