We reported earlier in a guinea pig model that exposure of 2-chloroethyl ethyl sulfide (CEES), a mustard gas analog, causes lung injury associated with the activation of tumor necrosis factor alpha (TNF-α), mitogen activated protein kinases (MAPK) signaling, and activator protein-1 (AP-1) transcription factor. Our earlier studies also revealed that antioxidant liposomes can be used as antidotes. Proinflammatory cytokines IL-1, IL-6, and TNF-α, either alone or in combination, can induce the activation of another group of transcription factors, namely SAF-1 (serum accelerator factor-1)/MAZ (Myc-associated zinc finger protein). Phosphorylation of SAF-1 via MAPK markedly increases its DNA-binding and transactivational potential. The objective of the present study was to investigate whether CEES exposure causes activation of IL-1β, IL-6, and SAF-1/MAZ and whether these effects can be prevented by antioxidant liposomes. A single dose (200 μL) of the antioxidant liposome mixture was administered intratracheally after 5 min of exposure of CEES (0.5 mg/kg). The animals were sacrificed either 1 h or 30 days after CEES exposure. CEES exposure caused an upregulation of proinflammatory cytokines IL-6 and IL-1β in the lung along with an increase in the activation of transcription factor SAF-1/MAZ. The antioxidant liposomes treatment significantly blocked the CEES-induced activation of IL-6, IL-1β, and SAF-1/MAZ. This might suggest that antioxidant liposomes might offer a potential therapeutic strategy against inflammatory diseases associated with activation of these bioactive molecules.

LPS pretreatment of human pro-monocytic THP-1 cells induces tolerance to secondary LPS stimulation with reduced TNFα production. However, secondary stimulation with heat-killed Staphylococcus aureus (HKSa) induces priming as evidenced by augmented TNFα production. The pro-inflammatory cytokine, IFNγ, also abolishes suppression of TNFα in LPS tolerance. The effect of LPS tolerance on HKSa and IFNγ-induced inflammatory mediator production is not well defined. We hypothesized that LPS, HKSa and IFNγ differentially regulate pro-inflammatory mediators and chemokine production in LPS-induced tolerance. THP-1 cells were pretreated for 24h with LPS (100ng/ml) or LPS (100ng/ml)+IFNγ (1μg/ml). Cells were subsequently stimulated with LPS or HKSa (10μg/ml) for 24h. The production of the cytokines TNFα, IL-6, IL-1β, and GMCSF and the chemokine IL-8 were measured in supernatants. LPS and HKSa stimulated TNFα (3070±711pg/ml and 217±9pg/ml, respectively) and IL-6 (237±8.9pg/ml and 56.2±2.9pg/ml, p<0.05, n=3, respectively) in control cells compared to basal levels (<25pg/ml). LPS induced tolerance to secondary LPS stimulation as evidenced by a 90% (p<0.05, n=3) reduction in TNFα. However, LPS pretreatment induced priming to HKSa as demonstrated by increased TNFα (2.7 fold, from 217 to 580pg/ml, p<0.05, n=3). In contrast to suppressed TNFα, IL-6 production was augmented to secondary LPS stimulation (9 fold, from 237 to 2076pg/ml, p<0.01, n=3) and also primed to HKSa stimulation (62 fold, from 56 to 3470pg/ml, p<0.01, n=3). LPS induced IL-8 production and to a lesser extent IL-1β and GMCSF. LPS pretreatment did not affect secondary LPS stimulated IL-8 or IL-1β, although HKSa stimulation augmented both mediators. In addition, IFNγ pretreatment reversed LPS tolerance as evidenced by increased TNFα levels while IL-6, IL-1β, and GMCSF levels were further augmented. However, IL-8 production was not affected by IFNγ. These data support our hypothesis of differential regulation of cytokines and chemokines in gram-negative- and gram-positive-induced inflammatory events. Such changes may have implications in the pathogenesis of polymicrobial sepsis.

Breast cancer is the second most common malignancy in the world, accounting for over 1.7 million new diagnoses and an estimated 500,000 deaths per year (1). Overexpression of the receptor tyrosine kinase ErbB2, also known as Her2 or Neu, occurs in over 30% of breast cancers and correlates with metastasis, poor prognosis, and decreased survival (1, 2). Although therapeutics targeting ErbB2 show clinical efficacy, many patients display no initial response or develop drug resistance over time (2). A deeper understanding of the molecular basis of ErbB2-driven tumorigenesis is thus required for the development of improved therapeutic strategies. In vitro experiments suggest that activation of the c-Jun NH2-terminal kinase (JNK) pathway, a mitogen-activated protein kinase pathway, promotes proliferation, cellular invasion, and stem cell expansion in ErbB2-driven breast cancer (3, 4). Furthermore, unpublished data from our lab using mammary epithelial cells expressing activated ErbB2 show that JNK is required for acinus formation in in vitro 3D cultures. In contrast to these studies showing a tumorigenic role for the JNK pathway, other data from our lab show that JNK loss results in accelerated breast tumor growth, suggesting a tumor suppressive role (5, 6). However, these studies were performed in p53 knockout mice with or without a Kras mutation, where the latter required extensive aging and genomic instability to occur before differences in tumor growth were observable. To date, limited in vivo studies exist to confirm the role of JNK in more biologically relevant breast tumor models, such as in ErbB2-mediated cancer, which accounts for over 30% of all human breast cancers. In addition, the molecular mechanisms by which JNK signaling promotes ErbB2-driven tumorigenesis remains poorly understood. To address the discrepancy in JNK function between the in vitro ErbB2-driven breast cancer data and the in vivo p53 knockout tumor data, I began the development of an in vivo murine model to confirm the role of JNK in ErbB2-driven breast cancer. This mouse model will also allow us to test a potential mechanism by which JNK regulates tumorigenesis. Studies show that ErbB2-mediated secretion of the inflammatory cytokine IL6 promotes transformation and tumor growth by activation of the STAT3 transcription factor, triggering an IL6/STAT3 autocrine signaling loop (7,8). A major regulator of Il6 gene expression includes activator protein 1 (AP-1), a transcription factor composed of downstream JNK targets in the Jun protein family (9). In vitro experiments using ErbB2-overexpressing mammary epithelial cell lines show that chemical inhibition of JNK suppresses secreted IL6 protein levels, supporting a role for the JNK pathway in IL6 regulation (7). Thus, I hypothesize that JNK drives ErbB2-driven breast cancer by promoting IL6-mediated tumor progression. Addressing this will increase our understanding of the role of JNK in ErbB2-driven breast cancer and reveal a potentially new mechanism by which JNK functions in tumor progression. Additionally, I began the development of a mouse model that will allow us to investigate the role of JNK in macrophage polarization as an alternative mechanism by which JNK regulates ErbB2-driven breast cancer. In addition to promoting STAT3-dependent tumor growth, IL6 can indirectly drive tumorigenesis by promoting expression of the IL4 receptor in macrophages, triggering STAT6-mediated macrophage polarization towards the pro-tumorigenic M2 phenotype (10, 11). Unlike classically activated M1 macrophages, which promote inflammation and anti-tumor immunity, alternatively activated M2 macrophages function in immunosuppression and metastasis and correlate with advanced stages of breast cancer (12, 13). Further evidence supporting a role for the JNK pathway in macrophage polarization includes a recent study suggesting that JunB, a downstream JNK target and component of the AP-1 complex, plays a crucial role in the induction of M2 macrophage polarization in human alveolar macrophages (13). I hypothesize that activation of the JNK signaling pathway induces IL6-dependent macrophage polarization towards the pro-tumorigenic M2 phenotype. Addressing this hypothesis will determine for the first time whether JNK functions in regulating macrophage polarization within the tumor microenvironment, offering a potentially new mechanism by which JNK can promote ErbB2-driven breast cancer. Determining the role of JNK in ErbB2-mediated breast cancer will have direct therapeutic relevance, as targeting JNK has the potential to inhibit ErbB2-driven breast cancer and other IL6-mediated diseases. Investigating the underlying mechanisms by which JNK functions in ErbB2-positive breast cancer can also offer new molecular targets and further contribute to effective drug design.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18684号 / 医博第3956号 / 新制||医||1007(附属図書館) / 31617 / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 伊達 洋至, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19930号 / 医博第4150号 / 新制||医||1017(附属図書館) / 33016 / 京都大学大学院医学研究科医学専攻 / (主査)教授 野田 亮, 教授 小川 誠司, 教授 高田 穣 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

HIV infection has been shown to predispose patients to accelerated development of heart disease. One mechanism for this pathology may involve endothelial activation either by HIV itself or by its secreted proteins, gp120 (a viral envelope protein) and tat (a protein that upregulates transcription of viral genes). We have studied the effects of gp120 and tat on signaling and production of inflammatory cytokines by Human Pulmonary Artery Endothelial Cells (HPAEC). HPAEC were stimulated at varying time points with combinations of gp120, tat, and monokines (IL-1β and TNFα). Cell lysate fractions were analyzed for MAP Kinase activity and NFκB activation, and culture supernatants were assayed for inflammatory cytokines (IL-6 and IL-8). The production of IL-6 and IL-8 was significantly enhanced by tat but not by gp120. Both gp120 and tat, however, induced significant morphological changes in HPAEC. The only synergy noted was between high levels of tat and TNFα acting on the production of IL-6. When HPAEC were stimulated with IL-1β and TNFα, peak phosphorylation of p38 MAP Kinase was found at 45 minutes, while NFκB was maximally activated at two hours. Both the ERK1,2 and p38 cascades of MAP Kinase were activated by tat, and an increase in NFκB phosphorylation and translocation were noted. We conclude that the HIV tat protein could be involved in inflammatory changes in endothelium leading to the accelerated development of heart disease in HIV patients.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

No description available.

No description available.