Cyclosporine inhibits the development of green fluorescent protein (GFP)-specific immune responses after transplantation of GFP-expressing hematopoietic repopulating cells in dogs.

Green fluorescent proteins (GFPs) have been widely used to monitor gene transfer and expression after lentiviral and oncoretroviral transduction of hematopoietic cells. Studies have shown a complete disappearance of GFP-containing cells after transplantation of GFP-transduced repopulating cells in nonhuman primates that was further shown to be mediated by transgene-specific immune responses. We wished to evaluate whether cyclosporine could prevent immune responses to GFP. We first determined whether an immune response to GFP was responsible for the disappearance of gene-modified cells in dogs. We performed immune assays in two dogs transplanted with lentivirally transduced CD34+ cells. Blood samples were obtained twice per week for up to 800 days and the GFP transgene product was measured by flow cytometry in blood leukocytes. Peripheral blood leukocytes were stimulated in vitro for 5 days, using a panel of GFP peptides. Intracellular levels of tumor necrosis factor alpha (TNF-alpha), measured by flow cytometry, and T cell proliferation after GFP peptide stimulation were measured. Dogs that exhibited a decrease in GFP marking developed potent immune responses in vitro to the transgene product GFP as shown by an increase in GFP-specific TNF-alpha production (p < 0.05) when compared with nontransplanted controls. T cells from dogs with low GFP marking exhibited a significant increase in proliferation in response to GFP peptide stimulation in vitro (p < 0.05). To study whether cyclosporine could inhibit the development of GFP-specific immune responses, we treated five dogs with cyclosporine after transplantation of GFP-transduced hematopoietic cells. Dogs treated with cyclosporine after hematopoietic stem cell transplantation showed stable GFP marking in blood leukocytes over 800 days. Our data suggest that cyclosporine prevents immunoactivation against transgene products after transplantation of GFP-transduced hematopoietic stem cells as indicated by stable GFP marking.

Quinolinic acid in vivo synthesis rates, extracellular concentrations, and intercompartmental distributions in normal and immune-activated brain as determined by multiple-isotope microdialysis.

Quinolinic acid (QUIN) kills neurons by activation of NMDA receptors that are accessed via the extracellular fluid (ECF). In vivo microdialysis was employed to quantify the dynamics of ECF QUIN levels. [(13)C7]QUIN was perfused through the probe for in vivo calibration to accurately quantify ECF QUIN concentrations. Osmotic pumps infused [(2H)3]QUIN subcutaneously to quantify blood contributions to ECF and tissue levels. Local QUIN production rates and influx and efflux rates across the blood-brain barrier were calculated from the extraction fraction of [(13)C7]QUIN, probe geometry, tissue diffusion coefficients, the extracellular volume fraction, and [(2)H3]QUIN/QUIN ratios in blood and dialysates. In normal brain, 85% of ECF QUIN levels (110 nM) originated from blood, whereas 59% of tissue homogenate QUIN (130 pmol/g) originated from local de novo synthesis. During systemic immune activation (intraperitoneal injection of endotoxin), blood QUIN levels increased (10.2-fold) and caused a rise in homogenate (10.8-fold) and ECF (18.5-fold) QUIN levels with an increase in the proportions of QUIN derived from blood. During CNS inflammation (local infusion of endotoxin), increases in brain homogenate (246-fold) and ECF (66-fold) QUIN levels occurred because of an increase in local synthesis rate (146-fold) and a reduction in efflux/influx ratio (by 53%). These results demonstrate that brain homogenate measures are a reflection of ECF concentrations, although there are quantitative differences in the values obtained. The mechanisms that maintain ECF QUIN levels at low values cannot do so when there are large increases in local brain synthesis or when there are large elevations in blood QUIN concentrations.

Quinolinic acid is extruded from the brain by a probenecid-sensitive carrier system: a quantitative analysis.

Although the neurotoxic tryptophan-kynurenine pathway metabolite quinolinic acid originates in brain by both local de novo synthesis and entry from blood, its concentrations in brain parenchyma, extracellular fluid, and CSF are normally below blood values. In the present study, an intraperitoneal injection of probenecid (400 mg/kg), an established inhibitor of acid metabolite transport in brain, into gerbils, increased quinolinic acid concentrations in striatal homogenates, CSF, serum, and homogenates of kidney and liver. Direct administration of probenecid (10 mM) into the brain compartment via an in vivo microdialysis probe implanted into the striatum also caused a progressive elevation in both quinolinic acid and homovanillic acid concentrations in the extracellular fluid compartment but was without effect on serum quinolinic acid levels. A model of microdialysis transport showed that the elevations in extracellular fluid quinolinic acid and homovanillic acid levels following intrastriatal application are consistent with probenecid block of a microvascular acid transport mechanism. We conclude that quinolinic acid in brain is maintained at concentrations below blood levels largely by active extrusion via a probenecid-sensitive carrier system.

Species heterogeneity between gerbils and rats: quinolinate production by microglia and astrocytes and accumulations in response to ischemic brain injury and systemic immune activation.

Quinolinic acid is an excitotoxic kynurenine pathway metabolite, the concentration of which increases in human brain during immune activation. The present study compared quinolinate responses to systemic and brain immune activation in gerbils and rats. Global cerebral ischemia in gerbils, but not rats, increased hippocampus indoleamine-2,3-dioxygenase activity and quinolinate levels 4 days postinjury. In a rat focal ischemia model, small increases in quinolinate concentrations occurred in infarcted regions on days 1, 3, and 7, although concentrations remained below serum values. In gerbils, systemic immune activation by an intraperitoneal injection of endotoxin (1 mg/kg of body weight) increased quinolinate levels in brain, blood, lung, liver, and spleen, with proportional increases in lung indoleamine-2,3-dioxygenase activity at 24 h postinjection. In rats, however, no significant quinolinate content changes occurred, whereas lung indoleamine-2,3-dioxygenase activity increased slightly. Gerbil, but not rat, brain microglia and peritoneal monocytes produced large quantities of [13C(6)]-quinolinate from L-[13C(6)]tryptophan. Gerbil astrocytes produced relatively small quantities of quinolinate, whereas rat astrocytes produced no detectable amounts. These results demonstrate that the limited capacity of rats to replicate elevations in brain and blood quinolinic acid levels in response to immune activation is attributable to blunted increases in local indoleamine-2,3-dioxygenase activity and a low capacity of microglia, astrocytes, and macrophages to convert L-tryptophan to quinolinate.