Suppression of kindled seizures by paracrine adenosine release from stem cell-derived brain implants.

PURPOSE: Stem cells and their derivatives have emerged as a promising tool for cell-based drug delivery because of (a) their unique ability to differentiate into various somatic cell types, (b) the virtually unlimited donor source for transplantation, and (c) the advantage of being amenable to a wide spectrum of genetic manipulations. Previously, adenosine-releasing embryonic stem (ES) cells have been generated by disruption of both alleles of adenosine kinase (Adk-/-). Lack of ADK did not compromise the cells' differentiation potential into embryoid bodies or glial precursor cells. The aim of the present study was to investigate the potential of differentiated Adk-/- ES cell progeny for seizure suppression by paracrine adenosine release. METHODS: To isolate paracrine effects of stem cell-derived implants from effects caused by network integration, ES cell-derived embryoid bodies and glial precursor cells were encapsulated into semipermeable polymer membranes and grafted into the lateral brain ventricles of kindled rats. RESULTS: While seizure activity in kindled rats with wild-type Adk+/+ implants remained unaltered, rats with adenosine-releasing Adk-/- ES cell-derived implants displayed transient protection from convulsive seizures and a profound reduction of afterdischarge activity in EEG recordings. Long-term seizure suppression was precluded by limited viability of the encapsulated cells. CONCLUSIONS: We thereby provide a proof-of-principle that Adk-/- ES cell-derived brain implants can suppress seizure activity by a paracrine mode of action. Adk-deficient stem cells therefore represent a potential tool for the treatment of epileptic disorders.

Astrogliosis in epilepsy leads to overexpression of adenosine kinase, resulting in seizure aggravation.

Adenosine kinase (ADK) is considered to be the key regulator of the brain's endogenous anticonvulsant, adenosine. In adult brain, ADK is primarily expressed in a subpopulation of astrocytes and striking upregulation of ADK in these cells has been associated with astrogliosis after kainic acid-induced status epilepticus (KASE) in the kainic acid mouse model of temporal lobe epilepsy. To investigate the causal relationship between KASE-induced astrogliosis, upregulation of ADK and seizure activity, we have developed a novel mouse model [the Adktm1(-/-)-Tg(UbiAdk) mouse] lacking the endogenous astrocytic enzyme due to a targeted disruption of the endogenous gene, but containing an Adk transgene under the control of a human ubiquitin promoter. Mutant Adktm1(-/-)-Tg(UbiAdk) mice were characterized by increased brain ADK activity and constitutive overexpression of transgenic ADK throughout the brain, with particularly high levels in hippocampal pyramidal neurons. This ADK overexpression was associated with increased baseline levels of locomotion. Most importantly, two-thirds of the mutant mice analysed exhibited spontaneous seizure activity in the hippocampus and cortex. This was the direct consequence of transgene expression, since this seizure activity could be prevented by systemic application of the ADK inhibitor 5-iodotubercidin. Intrahippocampal injection of kainate in the mutant mice resulted in astrogliosis to the same extent as that observed in wild-type mice despite the absence of endogenous astrocytic ADK. Therefore, KASE-induced upregulation of endogenous ADK in wild-type mice is a consequence of astrogliosis. However, seizures in kainic acid-injected mutants displayed increased intra-ictal spike frequency compared with wild-type mice, indicating that, once epilepsy is established, increased levels of ADK aggravate seizure severity. We therefore conclude that therapeutic strategies that augment the adenosine system after astrogliosis-induced upregulation of ADK constitute a neurochemical rationale for the prevention of seizures in epilepsy.

Seizure suppression and lack of adenosine A1 receptor desensitization after focal long-term delivery of adenosine by encapsulated myoblasts.

Adenosine is an important inhibitory modulator of brain activity. In a previous ex vivo gene therapy approach, local release of adenosine by encapsulated fibroblasts implanted into the vicinity of an epileptic focus, was sufficient to provide transient protection from seizures (Huber, A., Padrun, V., Deglon, N., Aebischer, P., Mohler, H., Boison, D., 2001. Grafts of adenosine-releasing cells suppress seizures in kindling epilepsy. Proc. Natl. Acad. Sci. U. S. A. 98, 7611-7616). Long-term seizure suppression beyond 2 weeks was precluded by limited life expectancy of the encapsulated fibroblasts. To study the feasibility for long-term seizure suppression by adenosine releasing brain implants, in the present contribution, mouse C2C12 myoblasts were engineered to release adenosine by genetic inactivation of adenosine kinase. After encapsulation, the myoblasts were grafted into the lateral brain ventricles of epileptic rats kindled in the hippocampus. While seizure activity in animals with wild-type implants remained unaltered, 1 week after grafting all rats with adenosine-releasing implants (n = 25) displayed complete protection from convulsive seizures and a corresponding reduction of afterdischarges in EEG-recordings. The duration of seizure suppression was maintained for a period of 3 weeks in 50% of the animals ranging to a maximum of 8 weeks in one animal. During the course of these experiments, adenosine A1 receptors remained responsive to selective agonists and antagonists indicating a lack of desensitization of A1 receptors after local long-term exposure to adenosine. Furthermore, local release of adenosine did not affect locomotor activity, whereas systemic application of the A1 agonist 2-chloro-N6-cyclopentyladenosine caused strong sedation. Thus, the local release of adenosine by cellular implants provides a feasible option for a potential side-effect free approach for the long-term treatment of focal epilepsies.

Curcumin differentially modulates mRNA profiles in Jurkat T and human peripheral blood mononuclear cells.

Curcumin, the yellow pigment of the rhizome of Curcuma longa is known to inhibit the transcription factors AP-1, Egr-1, NF-kappaB, c-myc and several important signaling kinases. We therefore investigated the differential effects of curcumin in concentation between 1.5 and 13.6 microM on gene expression in T Jurkat CD4(+) and human peripheral blood mononuclear cells (PBMCs). Relative quantification with reverse transcription real-time PCR (RT-rt-PCR) showed that low concentrations of curcumin significantly down-regulated mitogen-induced granulocyte macrophage colony stimulating factor (GM-CSF) mRNA (3- to 5-fold at 3 microM) in a dose- and time-dependent manner in both cell types. In comparison, the down-regulation of inducible nitric oxide (iNOS) mRNA levels was less pronounced, but interferon gamma (IFN-gamma) mRNA was dose-dependently up-regulated with curcumin concentrations up to 8.2 microM. Cyclin D1 mRNA expression was specifically inhibited in Jurkat T cells and stimulated PBMCs. The transcription factors NF-kappaB and NF-ATc were not affected in PBMCs. Interleukin-2 (IL-2), and-6 (IL-6) mRNAs levels were not influenced markedly by curcumin in stimulated PBMCs, but significantly reduced in stimulated Jurkat T cells. In addition, cytotoxic effects and down-regulation of mRNAs, including p65 and the house-keeping genes could only be measured in Jurkat T cells. These findings confirm previous reports on the anti-neoplastic potential of curcumin and show that this compound differentially modulates the expression profile of Th1 cells and PBMCs.

Relative quantification of mRNA levels in Jurkat T cells with RT-real time-PCR (RT-rt-PCR): new possibilities for the screening of anti-inflammatory and cytotoxic compounds.

PURPOSE: Quantification of the pro-inflammatory action of mitogens on mRNA levels of growth-related genes, transcription factors, and cytokines in T cells as markers for the screening of compounds with immunomodulatory, anti-inflammatory or cytotoxic potential. METHOD: A reverse transcription-real time-polymerase chain reaction assay with TaqMan probes was developed. Jurkat T cells were treated with cyclosporin A, hypericin, capsaicin, and catechin before phorbol 12-myristate 13-acetate stimulation, and their effects on the relative mRNA levels were determined. A cell viability assay was performed in parallel. RESULTS: Cyclosporin A and capsaicin were potent inhibitors of PMA-induced cytokine transcription. Cyclosporin A further targeted cyclin D1 transcription. Capsaicin exhibited no effects on the cell viability at low concentrations, whereas cyclosporin A did. Hypericin down-regulated nearly all investigated mRNAs, resulting in a strong time-dependent cytotoxicity. Catechin showed no effects on mRNA levels and cell viability. CONCLUSIONS: The inhibition of the up-regulation of mRNA levels of cytokines points to a specific anti-inflammatory potential of capsaicin. Hypericin showed no specific effects on the mRNA expression. The overall decrease of mRNA levels is probably an early indication of the strong cytotoxic effect observed after 48 h. Therefore, quantification of mRNA levels by reverse transcription-real time-polymerase chain reaction is, in combination with the monitoring of cell viability, a valuable tool to distinguish between specific immunomodulatory and cytotoxic effects in vitro.

Seizure suppression by adenosine A(2A) receptor activation in a rat model of audiogenic brainstem epilepsy.

Adenosine is known to suppress seizure activity mainly by activation of adenosine A(1) receptors. However, little is known about the potential involvement of other types of adenosine receptors in seizure suppression. It was now tested whether activation of adenosine A(2A) receptors would be effective in the suppression of generalized brainstem seizures. Genetically epilepsy-prone rats were intraperitoneally injected with increasing doses of the A(2A) receptor agonist, 5'-(N-cyclopropyl)-carboxamido-adenosine (CPCA), and, for comparison, with the A(1) receptor agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA). Both CPCA and CCPA were effective in suppressing generalized brainstem seizures with minimal effective concentrations of 2.5 and 1.5 mg/kg, respectively. Seizure suppression was maintained when CPCA was co-injected with the peripherally acting adenosine receptor antagonist 8-(p-sulphophenyl)theophylline, suggesting that central activation of A(2A) receptors effectively contributes to seizure suppression.