We have produced and begun characterizing a transgenic mouse model, p65fl/fl/LysMCre, that lacks canonical nuclear factor-kappaB (NF-kB) signaling (p65) in cells of the myeloid lineage, which includes macrophages. NF-kB pathway activity is very important in normal immune function, synaptic plasticity, and memory, and aberrant NF-kB activity is associated with autoimmune disease, and importantly, cancer. Macrophages can be present in very large numbers in a variety of cancers, and can lead to tumor progression through promotion of tumor inflammation, angiogenesis, invasion, and metastasis. This animal model will allow our group to pursue experiments involved in better understanding how stromal macrophages communicate with cancer cells through the NF-kB pathway, and how loss of canonical NF-kB signaling in cells of the myeloid lineage might weaken the tumor and make it more susceptible to standard treatments. Characterization of the model thus far reveals that p65 protein is indeed absent in macrophages derived from bone marrow monocytes, and that NF-kB signaling is altered when stimulated with lipopolysaccharide. We have just begun co-culture experiments with p65 deleted macrophages and glioma cells, and anticipate altered communication when compared to culture with control macrophages. Funding Source: Cancer Center Collaboration Grant

Cancer consists of malignant tumor cells as well as supporting, non-cancerous cells that make up the tumor stroma. Tumor-associated macrophages (TAMs), a critical component of the stroma, can be present in very large numbers in a variety of cancers, and can lead to tumor progression through promotion of tumor inflammation, angiogenesis, invasion, and metastasis. Canonical nuclear factor-kappaB (NF-κB) pathway activity is very important in normal immune function, synaptic plasticity, and memory, and aberrant NF-κB activity is associated with autoimmune disease, and importantly, cancer. Previous studies have been reported about the importance of tumor cell associated NF-κB signaling in cancers. As myeloid cell NF-κB signaling may also be important in promoting cancers, we have been utilizing the p65fl/fl/LysMCre transgenic animal model, which lacks p65 protein in cells of the myeloid lineage, to study the impact of myeloid cell derived NF-κB signaling in glioblastoma (GBM), an extremely aggressive brain cancer. This transgenic model has a very efficient deletion of p65 protein and drastically reduced NF-κB signaling in bone marrow derived macrophages (BMDMs), but brain residing microglia do not have significantly lower p65 levels as compared to control microglia. Even with this finding, p65fl/fl/LysMCre mice implanted with syngeneic GBM cells have significantly reduced GBM tumor burden than LysMCre control mice, as measured by magnetic resonance imaging. This result underscores the potential importance of bone marrow cells that migrate to the tumor site and significantly contribute to GBM growth. This work also indicates the potential benefits of targeting myeloid specific NF-κB signaling in GBM patients.

Consequences of pain include stimulation of some behaviors (e.g. reflexive withdrawal from stimuli), and depression of others (e.g. exercise, and work). Pain depressed behaviors are among the primary diagnostic and treatment concerns for physicians, but preclinical research has often explored pain stimulated behaviors. This discrepancy between basic research and clinical application may be one obstacle to the development of new pain treatments. In the present study, we modeled pain-related depression of behavior by observing nesting behavior in male ICR mice. Nest building is an innate mouse behavior that is reduced when the mouse is exposed to a pain stimulus. Pain-related depression of nesting is blocked by the clinically effective nonsteroidal anti-inflammatory drug (NSAID) ketoprofen. This project examines effects of monoamine uptake inhibitors with varying selectivity for serotonin (5HT), norepinephrine (NE) and dopamine (DA) on pain-related depression of nesting. Citalopram (5HT-selective), nisoxetine (NE-selective), milnacipran (mixed action, 5HT/NE-selective), and bupropion (DA-selective) were evaluated for their ability to block pain-related depression of nesting. Results show that the monoamine uptake inhibitors lacking significant dopamine had no effect on pain-depressed nesting. This finding is consistent with previous work suggesting that dopamine may be a key neurochemical target in the treatment of pain-related depression of behavior. Funding Source: Department of Psychological Sciences

Obesity often leads to hypertension. Previous work from my lab demonstrated that the adipocyte-derived hormone leptin reduces the ability of the aorta to contract in response to adrenergic stimulation, likely due to a decreased expression of the aorta alpha-adrenergic receptors. However, it is not known whether leptin decreases the expression of alpha-adrenergic receptors in such arteries like the mesenteric and renal arteries that play a key role in the control of blood pressure. To determine whether leptin decreases alpha-adrenergic receptor expression in renal and mesenteric arteries, I infused leptin (10ug/day) by implanting subcutaneous mini-pumps in five male C57bl/6 mice. Five mice did not receive leptin and served as controls. After seven days of treatment I euthanized nine mice as one mouse died. Renal and mesenteric arteries were taken from the mice and mRNa was extracted from the arteries. Reverse Transcription (RT) was completed in order to induce the transcription of mRNA into cDNA. After checking the concentration of cDNA, real-time PCR (qPCR) conducted on the arteries revealed high CT values for a1D-receptor concluding that leptin-mediated increases in sympathetic tone decreased a1D-receptor expression. This data is supportive of my hypothesis that leptin- decreases adrenergic receptor expression in renal and mesenteric arteries.

Hepatocellular carcinoma is the cancer of the liver cells that is developed over time by the evolution of pre-neoplastic lesions. Di-n-octylphthalate (DNOP) is a plasticizer used to keep plastics flexible. If mice are exposed to DNOP, it causes an increase in pre-neoplastic hepatic lesions. Previously, our group found that DNOP increased the expression of transforming growth factor β (tgf-β) in AML-12 cells. Because tgf-β induces an epithelial-to-mesenchymal transition (EMT) state in mouse hepatocyte in vitro, our goal was to study the extent to which DNOP induces an EMT state in mouse liver. Two antibodies were used: anti-albumin antibody (a hepatocyte marker), and anti-vimentin (a mesenchymal cell maker). We first treated AML-12 cells with 0.1 % DNOP for 24, 48 and 72 h. No changes in the expression of albumin was seen. Because the limited time of 72 h may not have allowed sufficient time for a change in the phenotype, mice were fed diet containing 0.1 % DNOP for a month. We found that DNOP decreased the levels of albumin, whereas increased the levels of vimentin. In conclusion, chronic consumption of DNOP induces an EMT state in mouse liver. This mechanism may be involved in formation of hepatic pre-neoplastic lesions.