Many inflammatory diseases are growing increasing common in the aging society of Taiwan. Inflammation cascades can cause diseases such as rheumatoid arthritis, osteoarthritis, chronic asthma, multiple sclerosis, and so on. The clinically used anti-inflammatory drugs have many side effects and are expensive. Therefore, it is imperative that we find alternatives to these drugs. Marine natural compounds offer great hope in the development of drugs for treating inflammatory diseases. In the present study, we found that Chao-10, which is a marine-derived compound isolated from Formosan soft coral, significantly inhibited the expression of the pro-inflammatory protein, inducible nitric oxide synthase (iNOS), in the lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophage cell line. We suggest that Chao-10 may serve as a potential new anti-inflammatory agent. However, the mechanism by which the anti-inflammatory effects of Chao-10 are mediated is yet unclear. Therefore, we performed two-dimensional electrophoresis (2-DE) to investigate the regulatory mechanism for the anti-inflammatory effect of Chao-10. We isolated some proteins that may be involved in the anti-inflammatory mechanism of Chao-10. In addition, we used immunoprecipitation to find that nucleophosmin (NPM) could interact with nuclear factor kappa B (NF-£eB). Therefore, we hypothesize that nucleophosminmay be involved in the regulation of NF-£eB to enhance the down-regulation of iNOS proteins. In summary, the anti-inflammatory effects of Chao-10 are probably mediated through the some other signaling pathway. Importantly, Chao-10 not only offers some new biomarkers of inflammation but also provides an encouraging outlook on therapeutic approaches.

In recent years, studies have increasingly recognized that many natural products with biological activity have been isolated from marine organisms, while the chemical structures are very different from those of land-based organisms. Therefore, the ocean is a natural drug source. Regarding drug screening, anti-inflammatory activity has become a key point, and many studies confirm that inflammation plays an important role in many human diseases. Many different compounds are now in the clinical evaluation stage. However, the inflammation-related diseases being closely linked, there is an urgent need to study the anti-inflammatory effects as well as screen the therapeutic drugs for research and development. In this study, we isolated and purified compounds from Formosan gorgonian (Briareum excavatum) and Formosan soft coral (Lobophytum sarcophytoides) and investigated biological activities. We confirmed that the natural compound Brei from B. excavatum and the compounds Sac-1 and Sac-2 from L. sarcophytoides produced significant inhibition of the proinflammatory proteins inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the lipopolysaccharide (LPS)-induced murine macrophages (RAW 264.7) cell model. We examined in vivo whether the B. excavatum Brei has anti-inflammatory and antinociceptive effects by using the carrageenan-induced inflammation model. Using the paw-edema assay, we performed several important investigations such as the plantar analgesia test, mechanical hyperalgesia test (allodynia), and weight-bearing analysis of animal behavior to evaluate the degree of pain and inflammation. Our results demonstrate that the natural product Brei can reduce paw-pad swelling, thermal hyperalgesia, threshold latency, and improve the affected limb in the carrageenan-induced inflammatory model. In the histopathology analysis, we showed that Brei significantly inhibited the aggregation and infiltration of inflammation-related blood cells and improved the inflammatory status of the tissues. Therefore, the marine natural compound Brei has anti-inflammatory activity and it can be used as a therapeutic compound for acute inflammation in the near future.

Activation of inducible nitric oxide synthase (iNOS) and oxidative stress have been shown to be associated with compromised cardiovascular function in streptozotocin (STZ)-induced type 1 diabetes. The aim of the project is to investigate cardiovascular abnormalities in a rat model of type 2 diabetes (Zucker diabetes fatty or ZDF rats) and two models of metabolic syndrome (fructose-fed rats and Zucker obese rats), and to provide direct evidence linking iNOS and oxidative stress to abnormal cardiovascular function in these disorders. Blood pressure, cardiac contractility, cardiac index, regional flow, vascular resistance and venous tone were measured in diseased as well as normal rats. Biochemical analyses such as activities of iNOS, immunostaining of iNOS and western-blot analysis of iNOS in the heart tissue were carried out. The results showed that cardiac contractile response to dobutamine was compromised in the ZDF rats, and this was associated with increased myocardial protein expression as well as activity of iNOS. The formation of peroxynitrite was increased in the heart tissue of the ZDF rats. Selective inhibition of iNOS by 1400W (N-3-aminomethyl-benzyl-acetamidine) did not alter responses to dobutamine in the control rats, but augmented the contractile effects of dobutamine in the diabetic rats. The regional blood flow was altered in the ZDF rats, and iNOS played a negligible role in regulating regional flow in the ZDF rats. Although venous response to noradrenaline was also altered in the Zucker obese rats, NOS may not be involved in venous tone regulation. Anti-oxidative treatment with N-acetylcysteine inhibited the development of insulin resistance, blood pressure elevation and the increase of 8-isoprostane formation in the fructose-fed rats. We conclude that heart function is compromised and regional blood flow is altered in the ZDF rats. Activation of iNOS plays an important role in suppressing heart dysfunction but does not affect regional blood flow. In Zucker obese rats with metabolic syndrome, iNOS may not be involved in changes of venous function. Oxidative stress is associated with both abnormality of heart dysfunction in type 2 diabetes (by formation of peroxynitrite due to iNOS activation) and development of hypertension and insulin resistance in metabolic syndrome.

No description available.

Activation of inducible nitric oxide synthase (iNOS) and oxidative stress have been shown to be associated with compromised cardiovascular function in streptozotocin (STZ)-induced type 1 diabetes. The aim of the project is to investigate cardiovascular abnormalities in a rat model of type 2 diabetes (Zucker diabetes fatty or ZDF rats) and two models of metabolic syndrome (fructose-fed rats and Zucker obese rats), and to provide direct evidence linking iNOS and oxidative stress to abnormal cardiovascular function in these disorders. Blood pressure, cardiac contractility, cardiac index, regional flow, vascular resistance and venous tone were measured in diseased as well as normal rats. Biochemical analyses such as activities of iNOS, immunostaining of iNOS and western-blot analysis of iNOS in the heart tissue were carried out. The results showed that cardiac contractile response to dobutamine was compromised in the ZDF rats, and this was associated with increased myocardial protein expression as well as activity of iNOS. The formation of peroxynitrite was increased in the heart tissue of the ZDF rats. Selective inhibition of iNOS by 1400W (N-3-aminomethyl-benzyl-acetamidine) did not alter responses to dobutamine in the control rats, but augmented the contractile effects of dobutamine in the diabetic rats. The regional blood flow was altered in the ZDF rats, and iNOS played a negligible role in regulating regional flow in the ZDF rats. Although venous response to noradrenaline was also altered in the Zucker obese rats, NOS may not be involved in venous tone regulation. Anti-oxidative treatment with N-acetylcysteine inhibited the development of insulin resistance, blood pressure elevation and the increase of 8-isoprostane formation in the fructose-fed rats. We conclude that heart function is compromised and regional blood flow is altered in the ZDF rats. Activation of iNOS plays an important role in suppressing heart dysfunction but does not affect regional blood flow. In Zucker obese rats with metabolic syndrome, iNOS may not be involved in changes of venous function. Oxidative stress is associated with both abnormality of heart dysfunction in type 2 diabetes (by formation of peroxynitrite due to iNOS activation) and development of hypertension and insulin resistance in metabolic syndrome.

Activation of inducible nitric oxide synthase (iNOS) and oxidative stress have been shown to be associated with compromised cardiovascular function in streptozotocin (STZ)-induced type 1 diabetes. The aim of the project is to investigate cardiovascular abnormalities in a rat model of type 2 diabetes (Zucker diabetes fatty or ZDF rats) and two models of metabolic syndrome (fructose-fed rats and Zucker obese rats), and to provide direct evidence linking iNOS and oxidative stress to abnormal cardiovascular function in these disorders. Blood pressure, cardiac contractility, cardiac index, regional flow, vascular resistance and venous tone were measured in diseased as well as normal rats. Biochemical analyses such as activities of iNOS, immunostaining of iNOS and western-blot analysis of iNOS in the heart tissue were carried out. The results showed that cardiac contractile response to dobutamine was compromised in the ZDF rats, and this was associated with increased myocardial protein expression as well as activity of iNOS. The formation of peroxynitrite was increased in the heart tissue of the ZDF rats. Selective inhibition of iNOS by 1400W (N-3-aminomethyl-benzyl-acetamidine) did not alter responses to dobutamine in the control rats, but augmented the contractile effects of dobutamine in the diabetic rats. The regional blood flow was altered in the ZDF rats, and iNOS played a negligible role in regulating regional flow in the ZDF rats. Although venous response to noradrenaline was also altered in the Zucker obese rats, NOS may not be involved in venous tone regulation. Anti-oxidative treatment with N-acetylcysteine inhibited the development of insulin resistance, blood pressure elevation and the increase of 8-isoprostane formation in the fructose-fed rats. We conclude that heart function is compromised and regional blood flow is altered in the ZDF rats. Activation of iNOS plays an important role in suppressing heart dysfunction but does not affect regional blood flow. In Zucker obese rats with metabolic syndrome, iNOS may not be involved in changes of venous function. Oxidative stress is associated with both abnormality of heart dysfunction in type 2 diabetes (by formation of peroxynitrite due to iNOS activation) and development of hypertension and insulin resistance in metabolic syndrome. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells identified in mice as Gr-1+CD11b+ cells with the ability to inhibit T cell function. MDSC are emerging as important regulators of T cell mediated immune responses. Current paradigm suggests that despite heterogeneity, all Gr-1+CD11b+ cells are suppressive when exposed to inflammatory stimuli. In vitro evaluation shows MDSC from multiple tissue sites have suppressive activity, and in vivo inhibition of MDSCenhances T cell function. However, the relative capacity of MDSC present at localized inflammatory sites or in peripheral tissues to suppress T cell responses in vivo has not been directly evaluated. Using a tissue specific acute inflammatory prostatitis model, we demonstrate that MDSC inhibition of CD8+ T-cell proliferation is restricted to the inflammatory site.Further, MDSC from inflammatory sites possess immediate capacity to inhibit T-cell function, whereas those isolated from peripheral tissues (spleens and liver) were not suppressive without activation of iNOS by exposure to IFN-_.Using two mouse models of prostate cancer, we extend these findings to thetumor micro-environment. During a chronic inflammatory response induced by tumorgrowth, we show Gr-1+CD11b+ cells from the tumor site possess immediate capacity toregulate effector T cell function whereas those from the spleen do not. In both tumormodels and in our prostatitis model, long term culture of activated T cells with splenicGr-1+CD11b+ cells converted precursor cells into functional MDSC during standard in vitro suppression assays. These data highlight the importance of MDSC in the prostate, and demonstrate the function of MDSC during a localized inflammatory response isrestricted to the site of an ongoing immune responseGrowing evidence suggests that prostatitis associated with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is mediated in part by the loss of T cell and B cell tolerance to prostate antigens. Clinical data demonstrates the presence of T cell proliferative responses to prostate auto-antigens in CP/CPPS patients. However, the mechanisms leading to this loss of tolerance are not clearly understood, largely because of a lack of available animal models. We report the development of a new mouse model for the study of chronic prostate inflammation (CPI), the Prostate Ovalbumin Expressing Transgenic-3 (POET-3) model. Adoptive transfer of antigen specific OT-I T cells induces CPI characterized by infiltration of exogenous (OT-I) and endogenous T cells into the prostate persisting as long as 45 days after transfer. In vitro and in vivo data demonstrate inflammation induced loss of T cell tolerance to prostate auto-antigens. Auto-antibody responses to prostate antigens were detected in POET-3 mice after induction of CPI. These data have important therapeutic implications for treatment of CPI.

No description available.

No description available.

Francisella tularensis, the causative agent of tularemia, is a potent human and animal pathogen. Initially upon infection of the host, intramacrophage proliferation of F. tularensis occurs but after activation of the acquired host immunity, the phagocytes become activated to kill the bacterium. In my thesis, I focused on mechanisms utilized by F. tularensis to survive intracellularly and on host mechanisms responsible for macrophage-mediated killing and control of infection. The F. tularensis-specific protein IglC has been previously shown to be essential to the intramacrophage proliferation and virulence of the bacterium in mice. By electron microscopy of macrophages infected with either the live vaccine strain of F. tularensis or an isogenic mutant, denoted ∆iglC, expression of IglC was found to be necessary for the bacterium to escape from the phagosome. IFN-g-activated macrophages significantly inhibited the escape of the live vaccine strain of F. tularensis from the phagosome. iNOS and phox generate NO and O2-, respectively. These molecules and their reaction products possess both bactericidal and immunoregulatory properties. We investigated the capability of IFN-g-activated peritoneal exudate cells from gene deficient iNOS-/- or p47phox-/- mice to control an intracellular F. tularensis LVS infection. iNOS was found to contribute significantly to the IFN-g induced killing, while phox contributed only to a minor extent. Unexpectedly, bacteria were eradicated even in the absence of both a functional phox and an active iNOS. The eradication was found to depend on ONOO-, the reaction product of NO and O2-, because addition of a decomposition catalyst of ONOO- completely inhibited the killing. Studies on iNOS-/- or p47phox-/- mice infected with F. tularensis LVS showed phox to be important during the first days of infection, a stage when iNOS seemed dispensable. Eventually, iNOS-/- mice died of the infection, suggesting a role of iNOS later in the course of infection. iNOS-/- mice exhibited elevated IFN-g serum levels and severe liver damage suggesting that the outcome of infection was at least in part related to an uncontrolled immune response. Several pathogenic bacteria express Cu,Zn-SOD, which in combination with other enzymes detoxifies reactive oxygen species produced by the host. A deletion mutant of F. tularensis LVS lacking the gene encoding Cu,Zn-SOD was attenuated at least 100-fold compared to LVS in mice. In peritoneal exudate cells from mice, Cu,Zn-SOD was found to be required for effective intramacrophage proliferation and, in mice, important for bacterial replication at the very early phase of infection. In summary, the most conspicuous findings were a capability of IFN-g activated macrophages to retain F. tularensis LVS in the phagosome, an essential role of ONOO- in intracellular killing of F. tularensis, and an importance of Cu,Zn-SOD to the virulence of F. tularensis LVS.