5-lipoxygenase expression and tepoxalin-induced cell death in squamous cell carcinomas in cats.

OBJECTIVE: To assess expression pattern and subcellular compartmentalization of 5-lipoxygenase in cutaneous, UV radiation-induced, and oral squamous cell carcinomas (SCCs) in cats and determine the effects of cyclooxygenase or 5-lipoxygenase inhibition on proliferation or apoptosis in a feline oral squamous cell carcinoma (SCCF1) cell line. SAMPLE: 60 archived paraffin-embedded samples of SCCs from 60 cats and SCCF1 cells. PROCEDURES: Retrospective immunohistochemical analysis of the archived samples of SCCs (20 cutaneous, 20 UV radiation-induced, and 20 oral tumors) was performed. Cell culture proliferation assays involving SCCF1 cells were performed, and tepoxalin-induced apoptosis and signaling were examined via western blotting and annexin V staining. RESULTS: Immunohistochemically, staining for 5-lipoxygenase was most frequently of greatest intensity in oral SCCs, whereas staining of cutaneous and UV radiation-induced lesions had less consistent 5-lipoxygenase expression. Exposure of SCCF1 cells to the 5-lipoxygenase inhibitor tepoxalin resulted in apoptosis; the effect appeared to be mediated via alteration of cell signaling rather than via suppression of lipid mediators that are typically produced as a result of 5-lipoxygenase activity. CONCLUSIONS AND CLINICAL RELEVANCE: In cats, expression of 5-lipoxygenase in SCCs appeared to differ depending on tumor location. The influence of tepoxalin-induced 5-lipoxygenase inhibition on a 5-lipoxygenase-expressing cell line coupled with the notable expression of 5-lipoxygenase in oral SCCs suggested that 5-lipoxygenase inhibition may have therapeutic benefits in affected cats. Although the safety of tepoxalin in cats has yet to be investigated, 5-lipoxygenase inhibitors should be evaluated for use as a potential treatment for SCCs in that species.

The effects of weight loss on adipokines and markers of inflammation in dogs.

Evidence suggests that adipose tissue-derived adipokines induce mild inflammation and may play a role in insulin resistance associated with diabetes. The present study was designed to examine a series of adipokines and markers of inflammation in dogs before and after a successful weight loss. The study included fasting serum samples from twenty-five dogs before and after a weight-loss programme. Serum C-reactive protein (CRP) and monocyte chemoattractant protein-1 (MCP-1) were measured as indicators of chronic inflammation, while serum adipokines including total adiponectin, high-molecular-weight (HMW) adiponectin, resistin and leptin were also examined. Medians for CRP (before, 10.0 (interquartile range 5.4-15.0) µg/ml; after, 5.6 (interquartile range 3.8-7.0) µg/ml) and MCP-1 (before, 212 (interquartile range 157-288) ng/ml; after, 185 (interquartile range 143-215) ng/ml) decreased significantly after weight loss. Medians for resistin showed a mild, yet significant reduction (before, 67.1 (interquartile range 44.4-88.5) pg/ml; after, 60.5 (interquartile range 32.3-67.1) pg/ml), while leptin showed a dramatic decrease after weight loss (before, 18.9 (interquartile range 10.8-35.4) ng/ml; after, 6.6 (interquartile range 3.9-10.2) ng/ml). Serum total adiponectin and HMW adiponectin were unchanged on all analyses performed. These data suggest that weight loss can decrease chronic inflammation; however, the clinical implications of this decrease are not well elucidated in dogs. Surprisingly, there was no increase in total or HMW serum adiponectin after weight loss, as observed previously in human subjects. The lack of change in total and HMW adiponectin might explain why insulin resistance and type 2 diabetes are less prevalent in obese dogs when compared with humans and cats.

NADPH oxidase limits innate immune responses in the lungs in mice.

BACKGROUND: Chronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide anion and downstream reactive oxidant intermediates (ROIs), is characterized by recurrent bacterial and fungal infections and by excessive inflammation (e.g., inflammatory bowel disease). The mechanisms by which NADPH oxidase regulates inflammation are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We found that NADPH oxidase restrains inflammation by modulating redox-sensitive innate immune pathways. When challenged with either intratracheal zymosan or LPS, NADPH oxidase-deficient p47(phox-/-) mice and gp91(phox)-deficient mice developed exaggerated and progressive lung inflammation, augmented NF-kappaB activation, and elevated downstream pro-inflammatory cytokines (TNF-alpha, IL-17, and G-CSF) compared to wildtype mice. Replacement of functional NADPH oxidase in bone marrow-derived cells restored the normal lung inflammatory response. Studies in vivo and in isolated macrophages demonstrated that in the absence of functional NADPH oxidase, zymosan failed to activate Nrf2, a key redox-sensitive anti-inflammatory regulator. The triterpenoid, CDDO-Im, activated Nrf2 independently of NADPH oxidase and reduced zymosan-induced lung inflammation in CGD mice. Consistent with these findings, zymosan-treated peripheral blood mononuclear cells from X-linked CGD patients showed impaired Nrf2 activity and increased NF-kappaB activation. CONCLUSIONS/SIGNIFICANCE: These studies support a model in which NADPH oxidase-dependent, redox-mediated signaling is critical for termination of lung inflammation and suggest new potential therapeutic targets for CGD.

Modeling the combination of amphotericin B, micafungin, and nikkomycin Z against Aspergillus fumigatus in vitro using a novel response surface paradigm.

Response surface methods for the study of multiple-agent interaction allow one to model all of the information present in full concentration-effect data sets and to visualize and quantify local regions of synergy, additivity, and antagonism. In randomized wells of 96-well plates, Aspergillus fumigatus was exposed to various combinations of amphotericin B, micafungin, and nikkomycin Z. The experimental design was comprised of 91 different fixed-ratio mixtures, all performed in quintuplicate. After 24 h of drug exposure, drug effect on fungal viability was assessed using the tetrazolium salt 2,3-bis {2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium-hydroxide} (XTT) assay. First, we modeled each fixed-ratio combination alone using the four-parameter Hill concentration-effect model. Then, we modeled each parameter, including the 50% inhibitory concentration (IC(50)) effect, versus the proportion of each agent using constrained polynomials. Finally, we modeled the three-agent response surface overall. The overall four-dimensional response surface was complex, but it can be explained in detail both analytically and graphically. The grand model that fit the best included complex polynomial equations for the slope parameter m and the combination index (equivalent to the IC(50) for a fixed-ratio concentration, but with concentrations normalized by the respective IC(50)s of the drugs alone). There was a large region of synergy, mostly at the nikkomycin Z/micafungin edge of the ternary plots for equal normalized proportions of each drug and extending into the center of the plots. Applying this response surface method to a huge data set for a three-antifungal-agent combination is novel. This new paradigm has the potential to significantly advance the field of combination antifungal pharmacology.