Clinical trials in amyotrophic lateral sclerosis: the tenuous past and the promising future

The past decade of research in amyotrophic lateral sclerosis has contributed to a greater understanding of the disease process, the development of relevant animal models, and the identification of several therapeutic approaches that may delay disease progression. Completed and ongoing clinical trials and the process of selecting drugs for clinical trials are presented.     

Seasonal variations of the beta-endorphin neuronal system in the mediobasal hypothalamus of the jerboa (Jaculus orientalis)

The distribution of neurons expressing beta-endorphin immunoreactivity was explored in the brain of adult jerboa during two distinct periods characterizing its reproductive cycle. A large presence of cell bodies displaying beta-endorphin immunoreactivity occured within different parts of the mediobasal hypothalamus along its rostrocaudal extent, from the retrochiasmatic area to the posterior arcuate nucleus. Quantitatively, the highest density of immunoreactive beta-endorphin neurons was noted at the medial level of the arcuate nucleus. Furthermore, a seasonal study showed that the number of IR-beta-endorphin neurons was highest in the anterior portion of the arcuate nucleus of jerboas sacrificed in autumn as compared to those sacrificed during spring-summer. Quantitatively, the number of beta-endorphin containing neurons in autumn was 200% in comparison to that found in spring-summer. These results suggest that beta-endorphin containing neuronal population especially localized in the anterior part of arcuate nucleus, exerts an inhibitory influence on the GnRH neurosecretory system in the jerboa, notably in autumn, probably via an increasing expression of its products. The results provide morphofunctional arguments in favour of inhibitory opioid control of GnRH neurons activity and hence the neuroendocrine events regulating reproduction in jerboa.     

Do platelet apoptosis, activation, aggregation, lipid peroxidation and platelet-leukocyte aggregate formation occur simultaneously in hyperlipidemia?

OBJECTIVES: The circulating lipoproteins may cause some abnormalities in platelet composition and function in hypercholesterolemia. The aim of this study was to investigate whether platelet apoptosis, platelet activation, platelet aggregation, platelet-leukocyte aggregate (PLA) formation and lipid peroxidation occur simultaneously in hyperlipidemia. DESIGN AND METHODS: Expression of GpIIb/IIIa (CD41a), P-selectin (CD62-P), platelet-bound fibrinogen (antifibrinogen), platelet membrane phosphatidylserine (PS), platelet-monocyte aggregates (mono-PLA) and platelet-neutrophil aggregates (neut-PLA) was measured in eight hyperlipidemic and eight normal subjects using flow cytometry. ADP (10 microM) was used to activate platelets. Furthermore, ADP induced platelet aggregation responses, platelet malondialdehyde (MDA) and glutathione (GSH) levels were determined. RESULTS: Before platelet activation, platelet CD62-P, antifibrinogen, annexin-V, mono-PLA, neut-PLA and platelet MDA levels as well as platelet aggregation responses in the hyperlipidemics were significantly higher than those in the controls (P<0.01, P<0.01, P<0.01, P<0.001, P<0.001, P<0.01, P<0.001, respectively), whereas GpIIb/IIIa expression and GSH levels were not different significantly (P > 0.05). In the control group, CD62-P, antifibrinogen and annexin-V levels increased significantly after ADP activation (P<0.05, P<0.05, P<0.01, respectively). In hyperlipidemic subjects, annexin-V expression increased significantly after activation (P<0.01), whereas expression of GpIIb/IIIa, CD62-P and antifibrinogen remained unchanged (P>0.05). The levels of total cholesterol (T-CHO), low density lipoprotein cholesterol (LDL-C), serum fibrinogen (S-FGN) and high density lipoprotein cholesterol (HDL-C) in patients were found to be correlated with platelet CD62-P, antifibrinogen, annexin-V, mono-PLA and MDA. CONCLUSIONS: In conclusion, it seems that in hyperlipidemia, some platelets are in an activated state in circulation, and that increased lipid peroxidation, early apoptosis, platelet-leukocytes aggregate formation and platelet aggregation altogether accompany this process.     

Effect of pupillary dilation on retinal nerve fiber layer thickness measurements using optical coherence tomography

PURPOSE: To evaluate the effect of pupillary dilation on retinal nerve fiber layer thickness (RNFL) measurements using optical coherence tomography (OCT-3). METHODS: Randomly chosen eyes of healthy individuals were scanned before and after pupillary dilation by two trained operators (R.G.O., R.V.) using OCT-3 (Carl Zeiss Meditec, Inc., Dublin, CA). Fast and regular RNFL (256 A-scans) OCT-3 protocols (software version A1.1) were used in each scanning session. RNFL thickness measurements before and after dilation were compared. RESULTS: Ten eyes of 10 subjects (6 females, 4 males) were enrolled. Mean age was 32.0 +/- 11.2 years (range, 21 to 52 years). Mean pupillary diameter before and after dilation was 2.9 +/- 0.6 mm and 7.6 +/- 0.8 mm, respectively (P < 0.0001, paired t-test). There was no significant difference in RNFL thickness measurements before and after dilation using both fast and regular RNFL protocols (P > or = 0.05 for all comparisons, paired t-test). Mean coefficients of variation for mean RNFL thickness measurements were 15.3% before and 13.7% after dilation for operator 1; and 10.8% before and 12.7% after dilation for operator 2 for the fast RNFL protocol and 11.3% versus 10.4% and 12.9 versus 11.1%, respectively, for the regular RNFL protocol. CONCLUSION: Pupillary dilation is not necessary in all subjects to obtain reproducible RNFL thickness measurements using OCT-3.     

Axotomy-dependent and -independent synapse elimination in organ cultures of Wld(s) mutant mouse skeletal muscle

Progressive "dying back" neurodegenerative diseases are debilitating due to loss of connectivity after nerve terminal and axonal withdrawal, which impairs peripheral nerve function and leads ultimately to neuronal cell death. The mutant mouse (Wallerian degeneration slow; Wld(s)) provides an accessible model system to understand orthograde and retrograde degeneration, because in these mice axotomy induces slow, progressive withdrawal of nerve terminals from motor endplates. Axon degeneration itself is about 10 times slower than in wild-type mice. We describe an organ culture paradigm that permits direct observation of the progressive changes in morphology of neuromuscular junctions in Wld(s) mutant mice. Normal nerve terminal and motor endplate morphology were maintained at most Wld(s) neuromuscular junctions for up to 72 hr in vitro. At others, synaptic boutons were removed from postsynaptic junctional folds in piecemeal fashion, as observed in adults in vivo. By contrast, nerve terminals degenerated rapidly and synchronously in wild-type muscle cultures, resembling Wallerian degeneration in vivo. These observations confirm that in Wld(s) mice, axotomy triggers a mechanism of nerve-terminal withdrawal that seems qualitatively different from that in wild-type animals. The piecemeal dismantling of presynaptic terminals resembles that occurring during neonatal synapse elimination. Organ cultures of neonatal Wld(s) muscle maintained for 1-2 days in vitro also showed no evidence of synaptic terminal degeneration, but elimination of polyneuronal innervation progressed in vitro at approximately the same rate as in vivo. Taken together, the data suggest that both natural and axotomy-induced forms of synapse withdrawal may be accessible to continuous observation and analysis, in organ-cultures of Wld(S) mouse muscles. This offers several advantages over repeated visualization of synaptic remodeling that has thus far been possible only in vivo.     

Mutational analysis of the CYP1B1 gene in Pakistani primary congenital glaucoma patients: Identification of four known and a novel causative variant at the 3′ splice acceptor site of intron 2

Primary congenital glaucoma (PCG) causes blindness in early age. It has an autosomal recessive pattern of inheritance, hence is more prevalent in populations with frequent consanguineous marriages that occur in the Pakistani population. Mutations in the CYP1B1 gene are commonly associated with PCG. The aim of the present study was to identify genetic mutations in the CYP1B1 gene in PCG cases belonging to 38 Pakistani families. DNA was extracted using blood samples collected from all enrolled patients, their available unaffected family members and controls. Direct sequencing of the CYP1B1 gene revealed a novel 3' splice acceptor site causative variant segregating in an autosomal recessive manner in a large consanguineous family with four PCG‐affected individuals. The novel variant was not detected in 93 ethnically matched controls. Furthermore, four already reported mutations, including p.G61E, p.R355X, p.R368H, and p.R390H were also detected in patients belonging to nine different families. All identified causative variants were evaluated by computational programs, that is, SIFT, PolyPhen‐2, and MutationTaster. Pathogenicity of the novel splice site variant identified in this study was analyzed by Human Splicing Finder and MaxEntScan. Ten out of 38 families with PCG had the disease due to CYP1B1 mutations, suggesting CYP1B1 was contributing to PCG in these Pakistani patients. Identification of this novel 3' splice acceptor site variant in intron 2 is the first report for the CYP1B1 gene contributing to genetic heterogeneity of disease.     

Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria.

Molecular assays for monitoring sulfadoxine-pyrimethamine-resistant Plasmodium falciparum have not been implemented because of the genetic and statistical complexity of the parasite mutations that confer resistance and their relation to treatment outcomes. This study analyzed pretreatment dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genotypes and treatment outcomes in a double-blind, placebo-controlled trial of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment for uncomplicated P. falciparum malaria. Multiple logistic regression was used to identify mutations that were predictive of treatment failure and to identify interactions and confounding factors. Infections caused by parasites with 3 DHFR mutations and 2 DHPS mutations (the "quintuple mutant") were associated with sulfadoxine-pyrimethamine treatment failure but not with chlorproguanil-dapsone treatment failure. The presence of a single DHFR mutation (Arg-59) with a single DHPS mutation (Glu-540) accurately predicted the presence of the quintuple mutant. If this model is validated in other populations, it will finally be possible to use molecular markers for surveillance of antifolate-resistant P. falciparum malaria in Africa.