Kevin S. Kinney

Characterization of monocyte chemoattractant protein-1 expression following a kainate model of status epilepticus

Brain injury due to seizure induces a robust inflammatory response that involves multiple factors. Although the expression of chemokines has been identified as a part of this response, there are remaining questions about their relative contribution to seizure pathogenesis. To address this, we report the expression profile of the chemokine, monocyte chemoattractant protein-1 (MCP-1, CCL2), during kainate-induced seizure in the rat hippocampus. Furthermore, we compare MCP-1 expression to the temporal profile of blood-brain barrier (BBB) permeability and immune cell recruitment at the injury site, since both of these events have been linked to MCP-1. We find that BBB permeability increased prior to upregulation of MCP-1, while MCP-1 upregulation and immune cell recruitment occurred concurrently, 7-13 h after opening of the BBB. Our findings support the following conclusions: (1) BBB opening to large proteins does not require MCP-1 upregulation; (2) Leukocyte immigration is not sufficient to induce BBB opening to large proteins; (3) MCP-1 upregulation likely mediates recruitment of macrophages/microglia and granulocytes during seizure injury, thus warranting further investigation of this chemokine.

Sympathetic innervation of the amphibian spleen: developmental studies in Xenopus laevis.

Spleens from larval and adult South African clawed frogs (Xenopus laevis) were examined using sucrose-potassium phosphate-glyoxylic acid (SPG) histofluorescence for norepinephrine. Innervation of the larval Xenopus spleen is barely detectable at stage 54 and gradually increases during prometamorphosis (stage 57/58) until metamorphic climax (stage 66). This development of innervation late in the larval life of the animal was highly sensitive to environmental conditions and to rapidity at which development occurred. Prevention of overt metamorphosis by sodium perchlorate blockade prevented the development of noradrenergic (NA) splenic innervation in some, but not all, tadpoles examined. Depletion of T-lymphocytes by early larval thymectomy did not alter the kinetics or pattern of splenic NA innervation.

Immune alterations in three mouse strains following 2-deoxy-d-glucose administration

Using 2-deoxy-D-glucose (2-DG)-induced stress, our laboratory has developed studies to define stress effects on immune responses. Here, we report effects of increasing doses of 2-DG on the immune response of BALB/c, C57BL/6 and BDF(1) mice 2 h after three injections of 0 to 2000 mg/kg of 2-DG. Female 4- to 5-week-old mice were euthanized and blood and spleens were collected. A suspension of partially purified mature T splenocytes was obtained by negative selection using J11.d2 antibodies. Glucose and corticosterone levels were measured in the plasma of each mouse. Splenocyte and mature T splenocyte suspensions were tested in in vitro proliferation assays with or without concanavalin A. Splenocytes were analyzed for the following cell-surface markers: CD3, TCR alpha/beta, CD4, CD8 and major histocompatibility complex (MHC) Class II. Significant increases in blood glucose levels were observed in C57BL/6 and BALB/c strains with the highest 2-DG dose (p<0.05). Corticosterone levels were higher in BDF(1) mice and C57BL/6 mice following the administration of 1000 and 2000 mg/kg of 2-DG, respectively (p<0.01). In vitro proliferation of mature T splenocytes in the presence of concanavalin A was decreased in BDF(1) (p<0.05) but not in BALB/c and C57BL/6 mice. In addition, in BDF(1) mice the decrease was highly correlated with an increase of CD3+ and TCR alpha/beta+ cells in the spleen. These results demonstrated high variability in the response of different mouse strains to 2-DG-induced stress.

Efficacy and Safety of Orally/Sublingually, Intranasally, and Intraperitoneally Administered Recombinant Murine Interferon in the Treatment of Murine Encephalomyocarditis Virus

Interferons (IFN) have been shown to be effective in protecting animals against lethal viral infections when administered systemically in relatively high doses. Intraperitoneal (i.p.) injection of mice with encephalomyocarditis virus (EMCV) gives rise to a rapidly progressive fatal disease characterized by central nervous system involvement and encephalitis. IFN-alpha has been shown to be effective in protecting mice against lethal EMCV infection when given via parenteral and oral/sublingual routes. The current study was designed to explore the ability of orally/sublingually and intranasally (i.n.) administered IFN-alpha to treat mice infected with EMCV in support of a planned clinical trial to evaluate efficacy of oral IFN-alpha in human viral infections. The primary objective of the study was to determine the efficacy of recombinant murine IFN-alpha (rMuIFN-alpha) in the treatment of mice infected with 100 LD(50) EMCV following oral, i.n., and i.p. administration at doses of 20,000 and 100,000 IU. The results of the current experiment did not indicate protection from infection with EMCV in mice that received IFN by the i.n. or oral/sublingual routes. The negative controls, infection of mice with 100 LD(50) of EMCV followed by treatment with excipient via all three routes, resulted in death of nearly all mice, as expected. The positive control, treatment of EMCV-infected (100 LD(50)) mice with rMuIFN-alpha via the i.p. route, was successful in protecting a significant number of mice from death compared with matched controls. This study points out the need to determine the optimum conditions for administration of oral/sublingual or i.n. IFN to insure maximum efficacy against viral infections.

121. Febrile response in an ectothermic species upon exposure to cytokines

While the behavioral and fever-inducing effects of cytokines in mammals have been well studied, little is known about their effects in reptiles. We decided to investigate the effects of cytokines and determine which (if any) were responsible for behavioral fever in lizards. We injected Dipsosaurus dorsalis with lipopolysaccaride (LPS), interluekin-1 (IL-1) or interferon-gamma (IFN-g), while continuously monitoring their body temperatures utilizing iButton temperature dataloggers. While LPS induced significant fever responses in the desert iguanas, the cytokines IL-1 and IFN-g produced a hypothermic reaction (IL-1 in the afternoon and night, while IFN-g was only significantly decreased during the night hours). These dissimilar results suggest that other cytokines may be more closely involved in the fever response in desert iguanas such as was seen post-LPS. However, the possibility may exist that there was a dosing effect, or turkey-IL-1 was not sufficiently similar to any putative reptilian IL-1 to exert “normal” fever induction.

Preliminary investigation of the role of IL-1 in the behavioral fever response of the green iguana, Iguana iguana

In response to infection, several species of reptiles and amphibians spend more time in basking, leading to an elevated body temperature, a thermoregulatory response known as behavioral or emotional fever (Kluger et al., 1975). A similar response can be elicited by injection of purified lipopolysaccharide (LPS) or even by the stress of handling in some cases (Deen and Hutchinson, 2000, Bicego et al., 2002, Cabanac and Gosselin, 1993). In mammals, a febrile response to infection is mediated, at least in part, by the secretion of IL-1 (Hansen et al., 2001). IL-1 acts in part on the hypothalamus in the brain, leading to a change in the body’s thermostat, elevating body temperature through increased metabolic rate (Hill et al., 2004). Such a response is also seen to LPS injection in mammals. Given that ectotherms exhibit the same end effect of infection or LPS injection, it may be that IL-1 is mediating behavioral fever, but this has not yet been investigated. Our research is investigating the possibility that IL-1 also elicits a febrile response in ectotherms by monitoring the body temperature and behavior of juvenile green iguanas (Iguana iguana) following systemic injection of IL-1.

# 31 Effect of sympathetic denervation on antigen-specific antibody production in Xenopus laevis

McKrae virus. On days 2, 6, and 10 post infection (pi), the infected right trigeminal ganglia (TG) was excised and separated into single cell suspensions. The cell population from the infected TG was counted and identified using flow cytometric analysis. On days 4 and 8 p.i., the right infected TG was excised and total RNA isolated to examine gene expression of anti-viral cytokines. Results.Animals exposed to SDR showed increased numbers of monocytes/macrophages in the infected TG on days 2, 6, and 10 post-primary infection compared to the home caged control group (HCC) (p < .01). The infected TG in the SDR group also showed an increase in gene expression for IFN-a; on day 4 pi compared to HCC (p < .03). Conclusions. Social disruption enhances the number of infiltrating monocytes/macrophages in the TG of mice during HSV-1 infection, which may contribute to the observed increase in the gene expression of anti-viral cytokines, such as IFN-a. Because monocytes/macrophages play a large role in limiting HSV-1 spread during lytic infection, this enhanced innate immune response may thwart the development of recurrent HSV-1 infections by decreasing the number of neurons that are latently infected and thus have the potential to reactivate the virus. Further research is needed to support this hypothesis.