Ozone-induced enhancement of airway hyperreactivity in rhesus macaques: Effects of antioxidant treatment.

BACKGROUND: Ozone (O3) inhalation elicits airway inflammation and impairs treatment responsiveness in asthmatic patients. The underlying immune mechanisms have been difficult to study because of the lack of relevant experimental models. Rhesus macaques spontaneously have asthma and have a similar immune system to human subjects. OBJECTIVES: We sought to investigate mucosal immune changes after O3 inhalation in a clinically relevant nonhuman primate asthma model and to study the effects of an antioxidant synthetic lignan (synthetic secoisolariciresinol diglucoside [LGM2605]). METHODS: A cohort of macaques (n = 17) previously characterized with airway hyperreactivity (AHR) to methacholine was assessed (day 1). Macaques were treated (orally) with LGM2605 (25 mg/kg) or placebo twice per day for 7 days, exposed to 0.3 ppm O3 or air for 6 hours (on day 7), and studied 12 hours later (day 8). Lung function, blood and bronchoalveolar lavage (BAL) fluid immune cell profile, and bronchial brushing and blood cell mRNA expression were assessed. RESULTS: O3 induced significant BAL fluid neutrophilia and eosinophilia and increased AHR and expression of IL6 and IL25 mRNA in the airway epithelium together with increased BAL fluid group 2 innate lymphoid cell (ILC2s), CD1c+ myeloid dendritic cell, and CD4+ T-cell counts and diminished surfactant protein D expression. Although LGM2605 attenuated some of the immune and inflammatory changes, it completely abolished O3-induced AHR. CONCLUSION: ILC2s, CD1c+ myeloid dendritic cells, and CD4+ T cells are selectively involved in O3-induced asthma exacerbation. The inflammatory changes were partially prevented by antioxidant pretreatment with LGM2605, which had an unexpectedly disproportionate protective effect on AHR.

LGM2605 Reduces Space Radiation-Induced NLRP3 Inflammasome Activation and Damage in In Vitro Lung Vascular Networks.

Updated measurements of charged particle fluxes during the transit from Earth to Mars as well as on site measurements by Curiosity of Martian surface radiation fluxes identified potential health hazards associated with radiation exposure for human space missions. Designing mitigation strategies of radiation risks to astronauts is critical. We investigated radiation-induced endothelial cell damage and its mitigation by LGM2605, a radioprotector with antioxidant and free radical scavenging properties. We used an in vitro model of lung vascular networks (flow-adapted endothelial cells; FAECs), exposed to gamma rays, low/higher linear energy transfer (LET) protons (3⁻4 or 8⁻10 keV/µm, respectively), and mixed field radiation sources (gamma and protons), given at mission-relevant doses (0.25 gray (Gy)⁻1 Gy). We evaluated endothelial inflammatory phenotype, NLRP3 inflammasome activation, and oxidative cell injury. LGM2605 (100 µM) was added 30 min post radiation exposure and gene expression changes evaluated 24 h later. Radiation induced a robust increase in mRNA levels of antioxidant enzymes post 0.25 Gy and 0.5 Gy gamma radiation, which was significantly decreased by LGM2605. Intercellular cell adhesion molecule-1 (ICAM-1) and NOD-like receptor protein 3 (NLRP3) induction by individual or mixed-field exposures were also significantly blunted by LGM2605. We conclude that LGM2605 is a likely candidate to reduce tissue damage from space-relevant radiation exposure.

Macrophage migratory inhibitory factor promotes bladder cancer progression via increasing proliferation and angiogenesis.

Macrophage migratory inhibitory factor (MIF) is a proinflammatory cytokine shown to promote tumorigenesis. Using the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) model of bladder cancer, we previously showed that MIF knockout mice display decreased angiogenesis and invasion compared with wild-type. This study examines the role of MIF in bladder cancer via use of oral inhibitors of MIF. In vitro, high-grade bladder cancer cells were treated with recombinant human MIF +/- (rhMIF+/-) inhibitor. Measurements included cell counts, proliferation by (3)H-thymidine incorporation (TdR), extracellular signal-regulated kinase (ERK) phosphorylation by western blot analysis, messenger RNA (mRNA) expression by quantitative PCR and protein secretion by enzyme-linked immunosorbent assay. Treatment with rhMIF increased ERK phosphorylation, cell counts, TdR and mRNA expression and protein secretion of vascular endothelial growth factor, which were blocked by specific inhibitors of ERK and MIF. In vivo, 3-month-old male C57Bl/6 mice were given BBN for 22 and 16 weeks in study 1 and study 2, respectively. Mice (n = 8-10 per group) were gavaged with vehicle or doses of MIF inhibitors daily from weeks 16-22 in both studies. Average bladder weights, reflecting tumor mass, tumor stage/burden, mitotic rate and proliferation indices, and microvessel densities were reduced in inhibitor groups versus controls. In summary, MIF promotes bladder cancer via increasing cell proliferation and angiogenesis and oral inhibitors of MIF may prove useful in treatment of this disease.

Synthesis and structure-activity relationships of pyrido[3,2-b]pyrazin-3(4H)-ones and pteridin-7(8H)-ones as corticotropin-releasing factor-1 receptor antagonists.

Pyrido[3,2-b]pyrazin-3(4H)-ones and pteridin-7(8H)-ones were evaluated as corticotropin-releasing factor-1 receptor antagonists. The synthesis, SAR studies and pharmacokinetic evaluation of these analogs are described herein.

A small-molecule inhibitor of macrophage migration inhibitory factor for the treatment of inflammatory disease.

Multiple sclerosis (MS) is a chronic, debilitating disease of the central nervous system (CNS) characterized by demyelination and axon loss. The proinflammatory cytokine macrophage migration inhibitory factor (MIF) has been shown to be elevated in the cerebrospinal fluid of patients during relapses. The purpose of this study was to evaluate a new small-molecule inhibitor of MIF and its ability to reduce the severity of an animal model of MS, experimental autoimmune encephalomyelitis (EAE). We utilized 2 structurally related isoxazolines, which show in vitro inhibition of MIF tautomerase activity. We found that administration of an inhibitor of MIF to mice with established EAE immediately reduced the severity of clinical signs and expanded a population of regulatory T lymphocytes. We also noted that the inhibitor reduced relapses of disease in a relapsing/remitting model of EAE. An analysis of leukocyte migration into the brain revealed that administration of inhibitor reduced entry of these cells. No effects on inflammatory cytokine production or T-cell activation in the periphery were noted. From these studies, we conclude that a small-molecule inhibitor of MIF reduces the severity of EAE and prevents access of immune cells into the CNS, which could be of therapeutic relevance to MS.

Macrophage migration inhibitory factor is a therapeutic target in treatment of non-insulin-dependent diabetes mellitus.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in the pathogenesis of a variety of autoimmune inflammatory diseases. Here, we investigated the role of MIF in the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM) using MIF(-/-) mice and a mouse model of streptozotocin (STZ)-induced NIDDM. Following single injection of STZ, MIF(+/+) BALB/c mice showed a significant increase in blood glucose levels, developed polyuria, and succumbed to disease. In contrast, no such increase in blood glucose was observed in MIF(-/-) BALB/c mice treated with STZ. These mice produced significantly less inflammatory cytokines and resistin as compared with MIF(+/+) mice and failed to develop clinical disease. Finally, oral administration of a small-molecule MIF antagonist, CPSI-1306, to outbred ICR mice following induction of NIDDM significantly lowered blood glucose levels in the majority of animals, which was also associated with a significant reduction in the levels of the proinflammatory cytokines IL-6 and TNF-alpha in the sera. Taken together, these results demonstrate that MIF is involved in the pathogenesis of NIDDM and is a therapeutic target to treat this disease.

Inhibition of p38 mitogen-activated protein kinase pathway as prophylaxis of postoperative ileus in mice.

BACKGROUND & AIMS: Postoperative ileus, an iatrogenic complication of abdominal surgery, is mediated by severe inflammation of the tunica muscularis. Macrophages that reside in the muscularis have important roles in initiating the inflammation. We investigated whether activation of the p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase is involved in the genesis of postoperative ileus, and whether p38-MAPK inhibition by the macrophage-specific inhibitor semapimod prevents intestinal dysmotility. METHODS: Postoperative ileus was induced by intestinal manipulation of the small bowel in mice. Protein kinase phosphorylation was assessed by immunoblotting of muscularis externa preparations. Proinflammatory gene expression was quantified by real-time polymerase chain reaction. Myeloperoxidase histochemistry for neutrophils was performed in jejunal segments. Nitric oxide production was measured by Griess reaction in smooth-muscle organ culture supernatants. Jejunal contractility was assessed within an organ bath setup. Intestinal motility was analyzed by gastrointestinal and colonic transit measurements. RESULTS: High levels of p38-MAPK and stress-activated protein kinase phosphorylation were observed immediately after intestinal manipulation. Semapimod treatment led to a significant decrease of p38-MAPK phosphorylation in macrophages; proinflammatory gene expression of macrophage inflammatory protein-1alpha, interleukin-6, monocyte chemoattractant protein-1, and intercellular adhesion molecule-1; and neutrophil infiltration. Furthermore, semapimod completely abrogated nitric oxide production within the tunica muscularis. Subsequently, semapimod prevented the suppression of smooth muscle contractility and small intestinal and colonic motility after intestinal manipulation. CONCLUSION: A single preoperative semapimod administration prevents intestinal macrophage activation and subsequent gastrointestinal dysmotility induced by abdominal surgery. Semapimod inhibits p38-MAPK and nitric oxide production in macrophages, making it a promising strategy for prophylaxis of postoperative ileus.

MIF family proteins in genitourinary cancer: tumorigenic roles and therapeutic potential.

Genitourinary cancers encompass some of the most common solid tumours and have high rates of morbidity and mortality. Inflammation is associated with enhanced tumorigenesis, and a number of pro-inflammatory mediators, such as macrophage migration inhibitory factor (MIF), also promote tumorigenesis. Studies of the role of MIF (which largely functions via the type II transmembrane receptor CD74) in prostate, bladder and kidney cancers suggest that it is a pro-tumorigenic factor in genitourinary malignancy. Inhibiting MIF activity in cell culture and in preclinical animal models of genitourinary cancers reduces the phenotypic hallmarks of cancer, such as proliferation, angiogenesis and tumour aggressiveness, by downregulating signalling pathways such as those regulated by extracellular signal-regulated kinase (ERK), protein kinase B and p53, and MIF may also reverse immunosuppression. Progress has been made in our understanding of the role of MIF (and its family member D-dopachrome tautomerase (DDT)) in genitourinary cancers and how it can be therapeutically targeted.

MIF antagonist (CPSI-1306) protects against UVB-induced squamous cell carcinoma.

UNLABELLED: Macrophage migration inhibitory factor (MIF) is a homotrimeric proinflammatory cytokine implicated in chronic inflammatory diseases and malignancies, including cutaneous squamous cell carcinomas (SCC). To determine whether MIF inhibition could reduce UVB light-induced inflammation and squamous carcinogenesis, a small-molecule MIF inhibitor (CPSI-1306) was utilized that disrupts homotrimerization. To examine the effect of CPSI-1306 on acute UVB-induced skin changes, Skh-1 hairless mice were systemically treated with CPSI-1306 for 5 days before UVB exposure. In addition to decreasing skin thickness and myeloperoxidase (MPO) activity, CPSI-1306 pretreatment increased keratinocyte apoptosis and p53 expression, decreased proliferation and phosphohistone variant H2AX (γ-H2AX), and enhanced repair of cyclobutane pyrimidine dimers. To examine the effect of CPSI-1306 on squamous carcinogenesis, mice were exposed to UVB for 10 weeks, followed by CPSI-1306 treatment for 8 weeks. CPSI-1306 dramatically decreased the density of UVB-associated p53 foci in non-tumor-bearing skin while simultaneously decreasing the epidermal Ki67 proliferation index. In addition to slowing the rate of tumor development, CPSI-1306 decreased the average tumor burden per mouse. Although CPSI-1306-treated mice developed only papillomas, nearly a third of papillomas in vehicle-treated mice progressed to microinvasive SCC. Thus, MIF inhibition is a promising strategy for prevention of the deleterious cutaneous effects of acute and chronic UVB exposure. IMPLICATIONS: Macrophage migration inhibitory factor is a viable target for the prevention of UVB-induced cutaneous SSCs.

Differentiation of Hdm2-mediated p53 ubiquitination and Hdm2 autoubiquitination activity by small molecular weight inhibitors.

The oncoprotein hdm2 ubiquitinates p53, resulting in the rapid degradation of p53 through the ubiquitin (Ub)-proteasome pathway. Hdm2-mediated destabilization and inactivation of p53 are thought to play a critical role in a number of human cancers. We have used an in vitro enzyme assay, monitoring hdm2-catalyzed Ub transfer from preconjugated Ub-Ubc4 to p53, to identify small molecule inhibitors of this enzyme. Three chemically distinct types of inhibitors were identified this way, each with potency in the micromolar range. All three types of compounds display selective inhibition of hdm2 E3 ligase activity, with little or no effect on other Ub-using enzymes. Most strikingly, these compounds do not inhibit the autoubiquitination activity of hdm2. Steady-state analysis reveals that all three classes behave as simple reversible inhibitors of the enzyme and that they are noncompetitive with respect to both substrates, Ub-Ubc4 and p53. Studies of the effects of combinations of two inhibitory molecules on hdm2 activity indicate that the three types of compounds bind in a mutually exclusive fashion, suggesting a common binding site on hdm2 for all of these inhibitors. These compounds establish the feasibility of selectively blocking hdm2-mediated ubiquitination of p53 by small molecule inhibitors. Selective inhibitors of hdm2 E3 ligase activity could provide a novel mechanism for the development of new chemotherapeutics for the treatment of human cancers.