Lymphocytes modulate innate immune responses and neuronal damage in experimental meningitis.

In bacterial meningitis, excessive immune responses carry significant potential for damage to brain tissue even after successful antibiotic therapy. Bacterial meningitis is regarded primarily as the domain of innate immunity, and the role of lymphocytes remains unclear. We studied the contribution of lymphocytes to acute inflammation and neurodegeneration in experimental Toll-like receptor 2-driven meningitis, comparing wild-type mice with RAG-1-deficient mice that have no mature T and B lymphocytes. At 24 h after intrathecal challenge with the synthetic bacterial lipopeptide Pam(3)CysSK(4), RAG-1-deficient mice displayed more pronounced clinical impairment and an increased concentration of neutrophils, reduced expression of interleukin-10 (IL-10) mRNA, and increased expression of CXCL1 mRNA in the cerebrospinal fluid. Conversely, neuronal loss in the dentate gyrus was reduced in RAG-1-deficient mice, and expression of IL-10, transforming growth factor ß and CCL2 mRNA by microglia was increased compared to wild-type mice. Adoptive transfer of wild-type lymphocytes reversed the enhanced meningeal inflammation and functional impairment observed in RAG-1-deficient mice. Our findings suggest compartment-specific effects of lymphocytes during acute bacterial meningitis, including attenuation of meningeal inflammation and shifting of microglial activation toward a more neurotoxic phenotype.

Absence of CCL2 is sufficient to restore hippocampal neurogenesis following cranial irradiation.

Cranial irradiation for the treatment of brain tumors causes a delayed and progressive cognitive decline that is pronounced in young patients. Dysregulation of neural stem and progenitor cells is thought to contribute to these effects by altering early childhood brain development. Earlier work has shown that irradiation creates a chronic neuroinflammatory state that severely and selectively impairs postnatal and adult neurogenesis. Here we show that irradiation induces a transient non-classical cytokine response with selective upregulation of CCL2/monocyte chemoattractant protein-1 (MCP-1). Absence of CCL2 signaling in the hours after irradiation is alone sufficient to attenuate chronic microglia activation and allow the recovery of neurogenesis in the weeks following irradiation. This identifies CCL2 signaling as a potential clinical target for moderating the long-term defects in neural stem cell function following cranial radiation in children.

In vivo near-infrared fluorescence imaging of matrix metalloproteinase activity after cerebral ischemia.

Matrix metalloproteinases (MMPs) have been implicated in the pathophysiology of cerebral ischemia. In this study, we explored whether MMP activity can be visualized by noninvasive near-infrared fluorescence (NIRF) imaging using an MMP-activatable probe in a mouse model of stroke. C57Bl6 mice were subjected to transient middle cerebral artery occlusion (MCAO) or sham operation. Noninvasive NIRF imaging was performed 24 h after probe injection, and target-to-background ratios (TBRs) between the two hemispheres were determined. TBRs were significantly higher in MCAO mice injected with the MMP-activatable probe than in sham-operated mice and in MCAO mice that were injected with the nonactivatable probe as controls. Treatment with an MMP inhibitor resulted in significantly lower TBRs and lesion volumes compared to injection of vehicle. To test the contribution of MMP-9 to the fluorescence signal, MMP9-deficient (MMP9(-/-)) mice and wild-type controls were subjected to MCAO of different durations to attain comparable lesion volumes. TBRs were significantly lower in MMP9(-/-) mice, suggesting a substantial contribution of MMP-9 activity to the signal. Our study shows that MMP activity after cerebral ischemia can be imaged noninvasively with NIRF using an MMP-activatable probe, which might be a useful tool to study MMP activity in the pathophysiology of the disease.

Live cell monitoring of mu-opioid receptor-mediated G-protein activation reveals strong biological activity of close morphine biosynthetic precursors.

G-protein activation by receptors is generally measured using (35)S-GTPgammaS binding assays in cell membranes and cannot be well assessed in intact cells. We have recently developed a fluorescence resonance energy transfer (FRET)-based approach to monitor G(i)-protein activation in living cells. Here we report that this technique can be used to determine structure-activity relationships of receptor agonists in intact cells. We have recently shown that morphine is biosynthesized de novo by mammals via a multistep pathway different from that in plants. However, the pharmacological properties of morphine precursors are poorly understood. Here, we directly monitored mu-opioid receptor (MOR)-mediated G(i)-protein activation in living cells by FRET and validated this method with classical GTPgammaS binding assays. Receptor binding studies and FRET measurements demonstrated that several (R)-configurated morphine precursors such as (R)-reticuline, salutaridine, salutaridinol, thebaine, and codeine were partial MOR agonists. Some closer precursors such as oripavine, codeinone, and morphinone activated G(i)-proteins as strongly as morphine, but with slightly lower potencies. The more distant the precursors were positioned in the pathway with respect to morphine, the less efficient and potent they were at MOR. Comparison of pharmacological properties of close morphine precursors and concentrations in which they occur in animal tissues suggests that they might activate MOR signaling under physiological conditions. Taken together, our data indicate that FRET-based assays of G-protein activation can serve to determine the abilities of compounds to activate G-protein signaling directly and in living cells.

Poppy alkaloid profiling by electrospray tandem mass spectrometry and electrospray FT-ICR mass spectrometry after [ring-13C6]-tyramine feeding.

Papaver alkaloids play a major role in medicine and pharmacy. In this study, [ring-(13)C(6)]-tyramine as a biogenetic precursor of these alkaloids was fed to Papaver somniferum seedlings. The alkaloid pattern was elucidated both by direct infusion high-resolution ESI-FT-ICR mass spectrometry and liquid chromatography/electrospray tandem mass spectrometry. Thus, based on this procedure, the structure of about 20 alkaloids displaying an incorporation of the labeled tyramine could be elucidated. These alkaloids belong to different classes, e.g. morphinan, benzylisoquinoline, protoberberine, benzo[c]phenanthridine, phthalide isoquinoline and protopine. The valuable information gained from the alkaloid profile demonstrates that the combination of these two spectrometric methods represents a powerful tool for evaluating biochemical pathways and facilitates the study of the flux of distant precursors into these natural products.

Analysis of benzylisoquinoline-type alkaloids by electrospray tandem mass spectrometry and atmospheric pressure photoionization.

Benzylisoquinoline alkaloids found in the Papaveraceae family play a major role in pharmaceutical biology. This is the first systematic study dealing with electrospray tandem mass spectrometry (ESI-MS/MS) of all benzylisoquinolines found as biogenetic precursors of morphinan alkaloids. Tandem mass spectral data are presented for norlaudanosoline, laudanosoline, 4'-O-methyl-norlaudanosoline, 6-O-methyl-norlaudanosoline, norcoclaurine, coclaurine, N-methylcoclaurine, N-methyl-3'-hydroxycoclaurine, N-methyl-3'-O-methylcoclaurine, norreticuline and reticuline. This study compares results obtained using an ion trap mass spectrometer with those obtained using a triple quadrupole one. The results highlight the differences of the tandem-in-time versus the tandem-in-space principle, often hampering the development of ESI-MS/MS libraries. Additionally, the use of the atmospheric pressure photoionisation technique for the analysis of such substances is discussed.

How human neuroblastoma cells make morphine.

Recently, our laboratory demonstrated that human neuroblastoma cells (SH-SY5Y) are capable of synthesizing morphine, the major active metabolite of opium poppy. Now our experiments are further substantiated by extending the biochemical studies to the entire morphine pathway in this human cell line. L-[1,2,3-13C3]- and [ring-2',5',6'-2H3]dopa showed high isotopic enrichment and incorporation in both the isoquinoline and the benzyl moiety of the endogenous morphine. [2,2-2H2]Dopamine, however, was exclusively incorporated only into the isoquinoline moiety. Neither the trioxygenated (R,S)-[1,3-13C2]norcoclaurine, the precursor of morphine in the poppy plant, nor (R)-[1,3,4-2H3]norlaudanosoline showed incorporation into endogenous morphine. However, (S)-[1,3,4-2H3]norlaudanosoline furnished a good isotopic enrichment and the loss of a single deuterium atom at the C-9 position of the morphine molecule, indicating that the change of configuration from (S)- to (R)-reticuline occurs via the intermediacy of 1,2-dehydroreticuline. Additional feeding experiments with potential morphinan precursors demonstrated substantial incorporation of [7-2H]salutaridinol, but not 7-[7-2H]episalutaridinol, and [7-2H,N-C2H3]oripavine, and [6-2H]codeine into morphine. Human morphine biosynthesis involves at least 19 chemical steps. For the most part, it is a reflection of the biosynthesis in opium poppy; however, there is a fundamental difference in the formation of the key intermediate (S)-reticuline: it proceeds via the tetraoxygenated initial isoquinoline alkaloid (S)-norlaudanosoline, whereas the plant morphine biosynthesis proceeds via the trioxygenated (S)-norcoclaurine. Following the plant biosynthetic pathway, (S)-reticuline undergoes a change of configuration at C-1 during its transformation to salutaridinol and thebaine. From thebaine, there is a bifurcate pathway leading to morphine proceeding via codeine or oripavine, in both plants and mammals.