Molecular imaging of cell therapy for gastroenterologic applications.

Stem cell therapy has appeared as a possible therapeutic alternative for numerous diseases. Furthermore, cancer stem cells are a focus of significant interest as they may allow for a better understanding of the genesis of different malignancies. The ultimate goal of stem cell therapeutics is to ensure the viability and functionality of the transplanted cells. Similarly, the ultimate goal of understanding cancer stem cells is to understand how they behave in the living subject. Until recently, the efficacy of stem cell therapies has been assessed by overall organ function recovery. Understanding the behavior and biology of stem cells directly in the living subject can also lead to therapy optimization. Thus, there is a critical need for reliable and accurate methods to understand stem cell biology in vivo. Recent advances in both imaging and molecular biology have enabled transplanted stem cells to be successfully monitored in the living subject. The use of molecular imaging modalities has the capability to answer these questions and may one day be translated to patients. In this review, we will discuss the potential imaging strategies and how they can be utilized, depending on the questions that need to be answered.

NADPH oxidase drives cytokine and neurotoxin release from microglia and macrophages in response to HIV-Tat.

Previous reports have shown that the human immunodeficiency virus (HIV) regulatory protein Tat has both pro-oxidant and pro-inflammatory properties, suggesting that Tat might contribute to the neurological complications of HIV. However, the intracellular mechanisms whereby Tat triggers free radical production and inflammation, and the relationship between Tat-induced free radicals and inflammatory reactions, are still subject to debate. The present study was undertaken to evaluate the specific effects of Tat on NADPH oxidase in microglia and macrophages, and to determine the specific role of NADPH oxidase in Tat-induced cytokine/chemokine release and neurotoxicity. Application of Tat to microglia or macrophages caused dose- and time-dependent increases in superoxide formation that were prevented by both pharmacologic NADPH oxidase inhibitors and by specific decoy peptides (gp91ds). Furthermore, inhibition of NADPH oxidase attenuated Tat-induced release of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF), and monocyte chemoattractant protein 1 (MCP-1), and decreased microglial-mediated neurotoxicity. Finally, macrophages derived from NADPH oxidase-deficient mice displayed reduced superoxide production, released lower levels of cytokines/chemokines, and induced less neurotoxicity in response to Tat compared to wild-type macrophages. Together, these data describe a specific and biologically significant signaling component of the macrophage/microglial response to Tat, and suggest the neuropathology associated with HIV infection might originate in part with Tat-induced activation of NADPH oxidase.

Gender and estrogen manipulation do not affect traumatic brain injury in mice.

As epidemiological data have suggested that female patients may have improved clinical prognoses following traumatic brain injury (TBI) compared to males, we designed experiments to determine the role of gender and estrogen in TBI-induced brain injury and inflammation in rodents. To this end, male and female C57Bl/6 mice were separated into the following four groups: intact males, intact females with vehicle supplementation, ovariectomized females with vehicle supplementation, and ovariectomized females with estrogen supplementation. All mice were subjected to a controlled cortical impact model of TBI, and cortical injury, hippocampal degeneration, microglial activation, and brain cytokine expression were analyzed after injury. Additionally, the spleens were harvested and cytokine release from cultured splenic cells was measured in response to specific stimuli. Data indicate that TBI-induced cortical and hippocampal injury, as well as injury-related microglial activation were not significantly affected by gender or estrogen manipulation. Conversely, brain levels of MCP-1 and IL-6 were significantly increased in males and intact females following TBI, but not in female mice that had been ovariectomized and supplemented with either estrogen or vehicle. Evaluation of splenic responses showed that the spleen was only moderately affected by TBI, and furthermore that spleens isolated from mice that had been given estrogen supplementation showed significantly higher release of the anti-inflammatory cytokine IL-4, regardless of the presence of absence of TBI. Overall, these data indicate that while estrogen can modulate immune responses, and indeed can predispose splenic responses towards and anti-inflammatory phenotype, these effects do not translate to decreased brain injury or inflammation following TBI in mice.

SOD1 overexpression alters ROS production and reduces neurotoxic inflammatory signaling in microglial cells.

Activation of the oxidative burst is one of the earliest biochemical events in microglial activation, but it is not understood yet how free radicals participate in inflammatory signaling. To determine the role that specific reactive oxygen species play in microglial activation, the levels of SOD1 were manipulated in N9 murine microglia. Stable overexpression of SOD1 caused significant decreases in superoxide and nitric oxide production, with concurrent increases in hydrogen peroxide following LPS. However, LPS-induced activation of NFkappaB, and release of TNFalpha and IL-6 were significantly attenuated in SOD1 overexpressing cells, as was the ability of microglia to induce toxicity in cultured neurons. Conversely, acute inhibition of SOD1 with disulfiram was associated with increased nitric oxide and cytokine release, and increased neurotoxicity. Together, these data suggest that superoxide radicals in microglia play important roles in directing redox-sensitive inflammatory signaling and initiating neurotoxic inflammation.

Estrogen and brain inflammation: effects on microglial expression of MHC, costimulatory molecules and cytokines.

To model the effects of estrogen on adaptive immunity in the brain, we examined the effects of 17beta-estradiol on microglial parameters related to antigen presentation and T cell activation. Specifically, the effects of 17beta-estradiol on basal and LPS-induced surface staining of Class I and II MHC, as well as CD40, CD80, CD86, CD152, CD28, CD8, CD11b, Fas, FasL, and also ERalpha and ERbeta, were examined in N9 microglial cells. Additionally, the effects of 17beta-estradiol on basal and LPS-induced release of cytokines (TNF-alpha, IFN-gamma, IL-2, IL-4, and IL-10) were determined. Data indicate that estrogen increases IL-10 while decreasing TNFalpha and IFNgamma release from resting and LPS-stimulated N9 cells. Additionally, LPS-induced surface staining of MHC Class I, CD40, and CD86 was significantly attenuated by estrogen pretreatment. The basal percentage of cells positive for MHC Class I and II, CD40, and CD152, Fas, and FasL was significantly decreased by estrogen exposure. However, CD8, CD86, CD11b, and CD28 were unaffected by estrogen, and CD80 cell surface staining significantly increased following estrogen exposure. Taken together, these data indicate that estrogen can significantly decrease components of adaptive immunity in microglial cells, and highlight the multi-faceted regulatory effects of estrogen on microglial parameters related to antigen presentation and T cell interaction.