Autologous hematopoietic cell transplantation following high-dose immunosuppressive therapy for advanced multiple sclerosis: long-term results.

The purpose of the study was to determine the long-term safety and effectiveness of high-dose immunosuppressive therapy (HDIT) followed by autologous hematopoietic cell transplantation (AHCT) in advanced multiple sclerosis (MS). TBI, CY and antithymocyte globulin were followed by transplantation of autologous, CD34-selected PBSCs. Neurological examinations, brain magnetic resonance imaging and cerebrospinal fluid (CSF) for oligoclonal bands (OCB) were serially evaluated. Patients (n=26, mean Expanded Disability Status Scale (EDSS)=7.0, 17 secondary progressive, 8 primary progressive, 1 relapsing/remitting) were followed for a median of 48 months after HDIT followed by AHCT. The 72-month probability of worsening ≥1.0 EDSS point was 0.52 (95% confidence interval, 0.30-0.75). Five patients had an EDSS at baseline of ≤6.0; four of them had not failed treatment at last study visit. OCB in CSF persisted with minor changes in the banding pattern. Four new or enhancing lesions were seen on MRI, all within 13 months of treatment. In this population with high baseline EDSS, a significant proportion of patients with advanced MS remained stable for as long as 7 years after transplant. Non-inflammatory events may have contributed to neurological worsening after treatment. HDIT/AHCT may be more effective in patients with less advanced relapsing/remitting MS.

Inhibition of glutamate-induced mitochondrial depolarization by tamoxifen in cultured neurons.

In central neurons, glutamate receptor activation causes massive calcium influx and induces a mitochondrial depolarization, which is partially blocked by cyclosporin A, suggesting a possible activation of the mitochondrial permeability transition pore (PTP) as a mechanism. It has been recently reported that tamoxifen (an antiestrogen chemotherapeutic agent) blocks the PTP in isolated liver mitochondria, similar to cyclosporin A. In this study, we tested whether tamoxifen inhibits the mitochondrial depolarization induced by glutamate receptor activation in intact cultured neurons loaded with the fluorescent dye 5,5',6,6'-tetrachloro-1,1',3, 3'-tetraethylbenzimidazolylcarbocyanine iodide. This dye reports disruptions in mitochondrial membrane potential, which can be caused by PTP activation. We found that glutamate (100 microM for 10 min) causes a robust mitochondrial depolarization that is partially inhibited by tamoxifen. The maximum inhibitory concentration of tamoxifen was 0.3 microM, with concentrations higher and lower than 0.3 microM being less effective. However, although tamoxifen (0.3 microM) blocked glutamate-induced mitochondrial depolarization, it did not inhibit glutamate-induced neuronal death, in contrast to the PTP inhibitor cyclosporin A. A relatively high concentration of tamoxifen (100 microM) caused mitochondrial depolarization itself and was neurotoxic. These data suggest that tamoxifen may be an inhibitor of the PTP in intact neurons. However, the lack of specificity of most PTP inhibitors, and the difficulty in measuring PTP in intact cells, preclude definite conclusions about the role of PTP in excitotoxic injury.

Methylmalonate toxicity in primary neuronal cultures.

Several inhibitors of mitochondrial complex II cause neuronal death in vivo and in vitro. The goal of the present work was to characterize in vitro the effects of malonate (a competitive blocker of the complex) which induces neuronal death in a pattern similar to that seen in striatum in Huntington's disease. Exposure of striatal and cortical cultures from embryonic rat brain for 24 h to methylmalonate, a compound which produces malonate intracellularly, led to a dose-dependent cell death. Methylmalonate (10 mM) caused >90% mortality of neurons although cortical cells were unexpectedly more vulnerable. Cell death was attenuated in a medium containing antioxidants. Further characterization revealed that DNA laddering could be detected after 3 h of treatment. Morphological observations (videomicroscopy and Hoechst staining) showed that both necrotic and apoptotic cell death occurred in parallel; apoptosis was more prevalent. A decrease in the ATP/ADP ratio was observed after 3 h of treatment with 10 mM methylmalonate. In striatal cultures it occurred concomitantly with a decline in GABA and a rise in aspartate content and the aspartate/glutamate ratio. Changes in ion concentrations were measured in similar cortical cultures from mouse brain. Neuronal [Na+]i increased while [K+]i and membrane potential decreased after 20 min of continuous incubation in 10 mM methylmalonate. These changes progressed with time, and a rise in [Ca2+]i was also observed after 1 h. The results demonstrate that malonate collapses cellular ion gradients, restoration of which imposes an additional load on the already compromised ATP-generation machinery. An early elevation in [Ca2+]i may trigger an increase in activity of proteases, lipases and endonucleases and production of free radicals and DNA damage which, ultimately, leads to cells death. The data also suggest that maturational and/or extrinsic factors are likely to be critical for the increased vulnerability of striatal neurons to mitochondrial inhibition in vivo.

Toxicity of dopamine to striatal neurons in vitro and potentiation of cell death by a mitochondrial inhibitor.

Intrastriatal injections of the mitochondrial toxins malonate and 3-nitropropionic acid produce selective cell death similar to that seen in transient ischemia and Huntington's disease. The extent of cell death can be attenuated by pharmacological or surgical blockade of cortical glutamatergic input. It is not known, however, if dopamine contributes to toxicity caused by inhibition of mitochondrial function. Exposure of primary striatal cultures to dopamine resulted in dose-dependent death of neurons. Addition of medium supplement containing free radical scavengers and antioxidants decreased neuronal loss. At high concentrations of the amine, cell death was predominantly apoptotic. Methyl malonate was used to inhibit activity of the mitochondrial respiratory chain. Neither methyl malonate (50 microM) nor dopamine (2.5 microM) caused significant toxicity when added individually to cultures, whereas simultaneous addition of both compounds killed 60% of neurons. Addition of antioxidants and free radical scavengers to the incubation medium prevented this cell death. Dopamine (up to 250 microM) did not alter the ATP/ADP ratio after a 6-h incubation. Methyl malonate, at 500 microM, reduced the ATP/ADP ratio by approximately 30% after 6 h; this decrease was not augmented by coincubation with 25 microM dopamine. Our results suggest that dopamine causes primarily apoptotic death of striatal neurons in culture without damaging cells by an early adverse action on oxidative phosphorylation. However, when combined with minimal inhibition of mitochondrial function, dopamine neurotoxicity is markedly enhanced.

Potential cost effectiveness of initial myocardial perfusion imaging for assessment of emergency department patients with chest pain.

Previous investigations have confirmed the diagnostic and predictive usefulness of initial single-photon emission computed tomography (SPECT) myocardial perfusion imaging using technetium-99m sestamibi in the evaluation of emergency department patients with chest pain. Patients with a normal SPECT perfusion scan performed during chest pain have an excellent short-term prognosis, and may be candidates for expeditious cardiac evaluation or outpatient management. However, there are limited data regarding the cost effectiveness of this technique. This analysis models the potential cost effectiveness of this procedure. In the current investigation we compared 2 model strategies for management of emergency department patients with typical chest pain and a normal or nondiagnostic electrocardiogram (ECG). In 1 model strategy, (the technetium-99m sestamibi SPECT myocardial perfusion imaging [SCAN] strategy), the decision whether to admit or discharge a patient from the emergency department is based on results of initial technetium-99m sestamibi SPECT myocardial imaging. Patients with normal scans are discharged; others are admitted. In the second model strategy, (the NO SCAN strategy), the decision whether or not to admit a patient is based on a combination of clinical and electrocardiographic variables. Patients with > or = 3 cardiac risk factors or an abnormal ECG are admitted; others are discharged. Adverse cardiac events were prospectively defined as cardiac death, nonfatal myocardial infarction, or the need for acute coronary intervention. Costs were assigned using data derived from 102 patients who underwent SPECT myocardial perfusion imaging and an additional 107 emergency department patients with ongoing chest pain who either underwent or were eligible for initial SPECT myocardial perfusion imaging. Mean (+/- SE) costs were highest among hospital admitted patients who experienced an adverse cardiac event ($21,375 +/- $2,733) and lowest in patients discharged from the emergency department ($715 +/- 71). Mean costs per patient of the SCAN strategy and NO SCAN strategy were $5,019 versus $6,051, respectively. These results were stable in a sensitivity analysis across a range of costs and predictive values. Thus, the SCAN model strategy for initial management of emergency department patients with typical ongoing angina and a normal or nondiagnostic ECG using initial myocardial perfusion imaging with technetium-99m sestamibi appears to be safe, accurate, and potentially cost effective. Validation of these preliminary retrospective observations will require further prospective investigation.

CAG trinucleotide RNA repeats interact with RNA-binding proteins.

Genes associated with several neurological diseases are characterized by the presence of an abnormally long trinucleotide repeat sequence. By way of example, Huntington's disease (HD), is characterized by selective neuronal degeneration associated with the expansion of a polyglutamine-encoding CAG tract. Normally, this CAG tract is comprised of 11-34 repeats, but in HD it is expanded to > 37 repeats in affected individuals. The mechanism by which CAG repeats cause neuronal degeneration is unknown, but it has been speculated that the expansion primarily causes abnormal protein functioning, which in turn causes HD pathology. Other mechanisms, however, have not been ruled out. Interactions between RNA and RNA-binding proteins have previously been shown to play a role in the expression of several eukaryotic genes. Herein, we report the association of cytoplasmic proteins with normal length and extended CAG repeats, using gel shift and UV crosslinking assays. Cytoplasmic protein extracts from several rat brain regions, including the striatum and cortex, sites of neuronal degeneration in HD, contain a 63-kD RNA-binding protein that specifically interacts with these CAG-repeat sequences. These protein-RNA interactions are dependent on the length of the CAG repeat, with longer repeats binding substantially more protein. Two CAG repeat-binding proteins are present in human cortex and striatum; one comigrates with the rat protein at 63 kD, while the other migrates at 49 kD. These data suggest mechanisms by which RNA-binding proteins may be involved in the pathological course of trinucleotide repeat-associated neurological diseases.

Evidence of excitotoxicity in the brain of the ornithine carbamoyltransferase deficient sparse fur mouse.

Ornithine carbamoyltransferase deficiency (OCTD) is the most common inborn error of urea synthesis. An X-linked disorder, OCTD males commonly present with hyperammonemic coma in the newborn period. There is a high rate of mortality and morbidity, with most survivors sustaining severe brain damage and resultant developmental disabilities. Although ammonia is presumed to be the principal neurotoxin, there is evidence that other neurochemical alterations may also be involved. The OCTD sparse fur (spf/Y) mouse has proven to be a useful model of this disease with similar metabolic and neurochemical alterations to those found in the human disease. In this study, the levels of the tryptophan derived excitotoxin quinolinic acid were examined in the brains of spf/Y mice. In addition, the neuropathology was examined using both light and electron microscopic approaches. Consistent with reports in children with urea cycle disorders, the levels of tryptophan and quinolinic acid were increased two-fold in various brain regions of the spf/Y mouse. Quinolinic acid, an agonist at the N-methyl-D-aspartate (NMDA) receptors, is known to produce selective cell loss in the striatum. We found a significant loss of medium spiny neurons and increased numbers of reactive oligodendroglia and microglia in the striatum of spf/Y mice. These neurochemical and neuropathological observations are consistent with an excitotoxic influence on brain injury in OCTD. It leads us to suggest that administration of NMDA receptor antagonists may ameliorate brain damage in children with inborn errors of urea synthesis.

The sparse fur mouse as a model for gene therapy in ornithine carbamoyltransferase deficiency.

The sparse fur (spf/Y) mouse was evaluated as a model for studying gene therapy in ornithine carbamoyltransferase deficiency (OCTD), the most common inborn error of urea synthesis. Previous studies have defined a number of biochemical characteristics of this animal model that are analogous to the human disease: OCTD in liver, elevated ammonium and glutamine, low citrulline and arginine in plasma, elevated urinary orotic acid excretion, neurochemical alterations and responsiveness to alternative pathway therapy. In this study, metabolic flux, survival, behavior and learning of these animals were examined in preparation for a trial of gene therapy. We found that, as has been previously reported, OCT activity in liver ranged from 10 to 20% of control. Yet, stable isotope studies using 15N ammonium chloride to follow ureagenesis in vivo showed 55% of normal urea synthetic capacity. This suggests that partial correction with gene therapy may be sufficient to normalize urea synthesis. Although it has been suggested that liver OCTD and its consequent metabolic effects normalize without treatment by adulthood in the spf/Y mouse, we did not find this to be the case. We documented that the spf/Y mouse had a markedly decreased lifespan (< 10% of normal) and remained runted throughout life. In terms of behavior, the spf/Y mice had evidence of decreased learning in a passive avoidance task that was not attributable to alterations in activity. These clearly definable metabolic and behavioral abnormalities suggest that the spf/Y mouse should prove a useful model for studying the efficacy of gene therapy in OCTD.