The safety and tolerability of single intravenous doses of lubeluzole (Prosynap) in healthy volunteers.

The safety and tolerability of single escalating doses of lubeluzole were evaluated in healthy male volunteers in 2 studies. In the first of 2 randomized, single-blind, placebo-controlled, dose-escalation studies, 6 subjects received single 30-minute infusions of 2.5, 5, and 10 mg of lubeluzole, and 2 additional subjects received placebo. In the second study 6 different subjects received a 1-hour infusion of 15 mg of lubeluzole, 5 of whom received the 20-mg dose, and 2 received 25 mg of lubeluzole. Two additional subjects received placebo. Small increases and decreases in PQ, QRS, QT, QTc, and QTm intervals were noted after infusion of all lubeluzole doses and placebo, however, these changes were within the normal ranges for these values except for the QTc for the 25-mg dose of lubeluzole. Significant prolongation of the QTc interval was observed at the end of the 1-hour infusion in both subjects receiving the 25-mg dose of lubeluzole. No clinically relevant changes in systolic time intervals, heart rate, blood pressure, and clinical laboratory values were noted in subjects receiving 2.5-25 mg of lubeluzole or placebo. Adverse experiences, predominantly lightheadedness and dizziness, were reported by subjects receiving doses of lubeluzole greater than or equal to 10 mg. Lubeluzole, administered as single intravenous doses of 2.5-15 mg, is safe and well tolerated in healthy male volunteers.

Safety and pharmacokinetics of the neuroprotective drug lubeluzole in patients with ischemic stroke.

A total of 22 patients with acute ischemic stroke participated in two randomized, single-masked, placebo-controlled studies that evaluated the safety and pharmacokinetics of single escalating intravenous doses of lubeluzole. The first dose of study medication in all patients was given within 6 hours of the first sign of stroke onset. In the first study, 6 patients received a single 1-hour intravenous infusion of 5 mg of lubeluzole; 4 of these patients received an additional 10-mg dose 3 to 4 days later. Two additional patients received placebo. In the second study, 4 patients received a single 1-hour infusion of 10 mg of lubeluzole, and 2 patients received placebo. After a safety evaluation of the second study, 6 additional patients received 15 mg of lubeluzole, and 2 other patients received placebo. Lubeluzole had no clinically relevant effects on any cardiovascular variable compared with placebo. The majority of adverse experiences were mild to moderate and resolved during treatment. No unexpected electroencephalogram abnormalities were observed, and no evidence of epileptiform discharges was found in any of the patients. At the end of the infusion, plasma lubeluzole concentrations decayed biphasically, with mean distribution half-lives of 46.3 to 101.0 minutes and mean terminal half-lives of 20.8 to 27.7 hours. Comparisons of the dose-normalized value of the individual plasma concentrations at the end of the infusion and the total area under the curve from time 0 to infinity suggested that lubeluzole exhibited linear kinetics over the dose range evaluated in patients with ischemic stroke. In the small number of patients studied, lubeluzole's favorable safety profile was demonstrated by the lack of clinically relevant effects on cardiovascular variables and by neurologic examination and clinical laboratory findings.

Hospital referral of stroke patients: a survey of attitudes in general practice, and consideration of entry times for clinical trials.

Two major issues in clinical trials in stroke are the criteria used for the selection of patients expected to benefit from the proposed treatment, and the entry time of those patients. We surveyed 507 Belgian general practitioners (GPs) on their opinions on referral of stroke patients to hospital and also on their actual referral behaviour. The feasibility of a 6-hour entry time was included in the investigation. Stroke is considered to require an urgent response: 88% of GPs visited the patient immediately on concluding that such an event had occurred. The mean time between the onset of the first clinical symptoms and the arrival of the GP at the patient's residence was about 30 minutes. Within 6 h of the insult, 95% of the patients referred to hospital had been admitted. Information on the GP's most recent stroke patient revealed that 72.4% of these stroke patients were admitted to hospital. Patients referred to hospital were significantly younger, had a significantly more severe stroke, and were significantly more likely to have had a first stroke and to have lived independently before the insult than patients not admitted to hospital. We think that Belgian GPs need to change their referral behaviour with respect to stroke patients and refer more of those who have suffered more mildly. There is every reason to be optimistic about this re-education, since the patients whom GPs do refer to hospital are referred rapidly enough to profit from a possibly efficacious treatment.

Reciprocal translocation between the proximal regions of the long arms of chromosomes 13 and 15 resulting in unbalanced offspring: characterization by fluorescence in situ hybridization and DNA analysis.

We describe two female siblings with similar clinical features consisting of hydrocephalus, scaphocephaly, hypotonia, mongoloid eye slant, blepharophimosis, micrognathia, supernumerary mouth frenula and mental retardation. Routine cytogenetic studies in the elder patient did not reveal any abnormality, and initially it was assumed that the syndrome had an autosomal recessive inheritance. However, a slightly larger chromosome 13 was seen in routine G-banded metaphases of the mother and the youngest of the two siblings. A shorter chromosome 15 was detected in the mother only. High resolution banding showed that the abnormal chromosome 13 contained an extra G-positive band at 13q12. The short chromosome 15 in the mother appeared to have a deletion of band q12. Fluorescence in situ hybridization using DNA markers specific to chromosomes 13 and 15 unequivocally showed that the mother was a carrier of a balanced reciprocal translocation t(13;15)(q12;q13), whereas the youngest sibling's karyotype was 46,XX,-13,+der(15)t(13;15)(q12;q13)mat, resulting in partial monosomy 13pter----q12 and partial trisomy 15pter----q13. The proband is thus trisomic for the critical region responsible for Prader-Willi syndrome and Angelman syndrome; this was confirmed by DNA analysis demonstrating one paternal and two maternal alleles from multiallelic marker loci mapping to 15q11-q13. This report illustrates the sensitivity and specificity offered by fluorescence in situ hybridization and its usefulness in the diagnosis and delineation of subtle chromosomal rearrangements.

Immunocytochemical detection of the growth-associated protein B-50 by newly characterized monoclonal antibodies in human brain and muscle.

The growth-associated protein B-50 also termed GAP-43, F1, pp46, P-57 and neuromodulin is a nervous tissue-specific protein kinase C (PKC) substrate that is considered to play a major role in neurite formation, regeneration, and neuroplasticity. We describe the isolation of seven mouse monoclonal antibodies (Mabs) directed against B-50. The Mabs are produced against the bovine B-50, selected by ELISA for cross-reactivity with its human counterpart, and evaluated on Western blots in comparison with the well-characterized affinity-purified rabbit polyclonal antibodies to rat-B-50. The Western blots show that the Mabs NM1, NM4, and NM6 recognize specifically the B-50 of bovine, human, and rat brain extract and the purified PKC phosphorylated and unphosphorylated rat B-50 isoforms. The Mabs NM2 and NM3 cross-react with bovine B-50 immunoreactive c-kinase substrate (BICKS), a protein sharing a 17 amino acid sequence homology with B-50. Two Mabs are useful for the detection of B-50 immunoreactivity in formalin-fixed human and rat brain tissues. In human specimen of the hippocampus, a characteristic neuropil distribution of B-50 is detected by the Mabs. In human muscle, Mabs reveal B-50 in nerve bundles and in axons at motor end plates. Thus, these Mabs are useful in investigating the function and localization of the B-50 protein.

Affinity purification of human tau proteins and the construction of a sensitive sandwich enzyme-linked immunosorbent assay for human tau detection.

Immunoaffinity chromatography with a monoclonal antibody produced against bovine tau protein was used to purify tau proteins from human brain. Fifty grams of brain tissue yielded approximately 2 mg of pure tau proteins. The affinity-purified human tau was used to produce a high-titered rabbit anti-human tau serum. The monoclonal anti-tau antibody and the polyclonal rabbit anti-tau serum were then used to construct a sandwich enzyme-linked immunosorbent assay for detection of human tau proteins, with a sensitivity of 1 ng/ml.

Specific monoclonal antibodies against normal microtubule-associated protein-2 (MAP2) epitopes present in Alzheimer pathological structures do not recognize paired helical filaments.

We have developed monoclonal antibodies that detect normal microtubule-associated protein-2 (MAP2) epitopes in routinely fixed, paraffin-embedded tissue. The somatodendritic distribution of MAP2 in bovine and human nervous tissue was confirmed with several of these antibodies. Furthermore, some of these antibodies immunohistochemically labeled certain pathological structures in Alzheimer brain, especially neurites in senile plaques. Electron microscopic observations, however, indicate that these MAP2 epitopes are not located in the Alzheimer paired helical filaments themselves, but in amorphous granular structures coexistent with them. While the pathological nature of these structures is undetermined, they may represent artefactual modifications of normal cytoskeletal components.

Monoclonal antibodies with selective specificity for Alzheimer Tau are directed against phosphatase-sensitive epitopes.

A modified form of the microtubule-associated protein Tau is the major component of the paired helical filaments (PHF) found in Alzheimer's disease. The characterization of these posttranslational Tau modifications is hindered by the lack of sufficient PHF-Tau-specific markers. Here we describe several monoclonal antibodies, prepared by immunization with PHF, two of which showed a selective specificity for PHF-Tau without cross-reactivity with normal Tau. Epitope recognition by these two monoclonals was sensitive to alkaline phosphatase treatment. In Western blotting these monoclonal antibodies reacted specifically with the abnormally phosphorylated epitopes on Alzheimer's disease-associated PHF-Tau. One of the new antibodies can be used for the construction of a sandwich enzyme-linked immunosorbent assay for the specific detection of PHF-Tau without cross-reactivity to normal Tau proteins.

Long term follow-up of multiple sclerosis by standardized, non-contrast-enhanced magnetic resonance imaging.

The usefulness of non-contrast-enhanced, standardized magnetic resonance imaging for the longterm follow-up of MS patients was evaluated in a retrospective study in 36 patients with clinically definite MS. All had remitting-relapsing diseases courses. Sixteen patients remained clinically stable during follow-up. Mean duration of follow-up was 22 months (SD: 11). A mean number of 3 MRI examinations was performed in each of the patients (SD: 1). Subclinical evolution was detected in 56% of the stable patients, indicating that clinical data alone are insufficient to assess disease activity. The relapsing patients showed significantly more and larger changes on MRI than stable patients (P less than 0.001), indicating that MRI is well suited as a follow-up parameter in conjunction with clinical data. The time courses of these quantitative changes and of the qualitative changes of putative MS lesions on MRI are discussed. It is concluded that MRI is a good indicator of global disease activity in multiple sclerosis patients, which makes MRI very useful for the evaluation of therapeutic trials.

Demonstration of a novel neurofilament associated antigen with the neurofibrillary pathology of Alzheimer and related diseases.

A monoclonal antibody, termed NFT200, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimer brain. The antigen to which NFT200 is directed was expressed in the paired helical filaments of NFT in sporadic and familial Alzheimer disease (AD), in the straight filaments of NFT in AD, progressive supranuclear palsy and of Pick bodies, and the NFT in several other conditions such as Parkinson-dementia complex of Guam and subacute sclerosing panencephalitis. Granulovacuolar degeneration of AD was also labeled with NFT200. Hirano bodies and amyloid deposits in AD, as well as Lewy bodies of idiopathic Parkinson disease lacked in the antigen. The NFT200-antigen was also expressed as a phosphatase-insensitive antigen in normal neurofilaments found in spinal cord and peripheral nerve axons but was absent from the perikaryal accumulation of neurofilaments induced by aluminum intoxication. Nevertheless, immunoblot studies failed to detect the NFT200 in isolated preparations of the neurofilament proteins, MAP-2, tau, ubiquitin or A4-amyloid peptide. The results indicate that the NFT200 monoclonal antibody is directed against a phosphatase-insensitive epitope of an axonal protein associated with neurofilaments but is labile to isolation and expressed as a stable epitope of a 200 kDa component of NFT.

Isolation of IgG1-secreting switch variants from IgM hybridomas produced after in vitro immunization.

IgG1-secreting variants have been isolated from three different IgM-secreting hybridomas, in two instances following in vitro immunization. The method used was based on sequential sublining in combination with selection by an IgG1-specific two-site ELISA system employing two different IgG1-specific polyclonal antisera. Idiotypic identity between the IgG1 variants and their respective IgM parent was demonstrated using syngeneic anti-idiotypic antisera. The antigen binding specificity in the IgG1 variants was also conserved. Isolation of naturally occurring IgG1 switch variants from IgM-secreting hybridomas that are produced after in vivo immunization offers a solution to the major disadvantages associated with the generation of IgM hybridomas.

Early-onset Alzheimer's disease in 2 large Belgian families.

Familial Alzheimer's disease (FAD) is a dominantly inherited condition that may present with an early onset, and myoclonus occurs frequently in the course of the disease. We report clinical and neuropathologic data on 2 large Belgian families with FAD in which we obtained 17 autopsies of the CNS. In family A, each of 11 autopsies had the typical neuropathologic features of Alzheimer's disease (AD), and there were a few cerebellar plaques in the molecular layer. In family B, in addition to the typical characteristics of AD in 6 autopsies, there were numerous amyloid plaques in the cortical cerebellar layers. In both families, we immunostained the amyloid deposits for the A4 protein, and they were negative for prion-associated protein immunoreactivity.

Assignment of the Charcot-Marie-Tooth neuropathy type 1 (CMT 1a) gene to 17p11.2-p12.

Charcot-Marie-Tooth disease type 1a (CMT 1a) is an autosomal dominant peripheral neuropathy linked to the DNA markers D17S58 and D17S71, located in the pericentromeric region of the chromosome 17p arm. We analyzed an extended 5-generation Belgian family, multiply affected with CMT 1a, for linkage with eight chromosome 17 markers. The results indicated that the CMT 1a mutation is localized in the chromosomal region 17p11.2-p12 between the marker D17S71 and the gene for myosin heavy polypeptide 2 of adult skeletal muscle.

A monoclonal antibody raised against Alzheimer cortex that specifically recognizes a subpopulation of microglial cells.

A monoclonal antibody (MAb), termed AMC30, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimer's brain. The antigen to which AMC30 is directed was expressed by microglial cells in senile plaques of Alzheimer's disease (AD). Microglia in the parenchyma surrounding brain tumors or infarctions, multinuclear giant cells, perivascular and parenchymal macrophages throughout the brain of AIDS patients were also labeled. Different non-nervous system lesions in which macrophages participate were also stained. Microglial cells in normal areas of the cortex or white matter were not labeled with MAb AMC30. The antigen to which AMC30 is directed was not detected in normal bone marrow, lymph nodes, lung, or spleen monocytes or macrophages. The epitope recognized by MAb AMC30 was present after formalin fixation and paraffin embedding. Our findings suggest that this MAb is directed against an antigen that is specifically expressed in a subpopulation of microglial cells and macrophages reactive to various pathological conditions.

Improved correlation of magnetic resonance imaging (MRI) with clinical status in multiple sclerosis (MS) by use of an extensive standardized imaging-protocol.

In a previous study we have shown that the sensitivity of magnetic resonance imaging (MRI) for the detection of multiple sclerosis (MS) lesions was improved significantly, especially in the infratentorial region, by use of an extensive standardized MRI-protocol consisting of sagittal T1, axial protondensity and axial T2, and sagittal protondensity and sagittal moderately T2-weighted images. The goal of the present study was to assess whether the clinical correlation of the visualized lesions had improved accordingly. Using a scoring system based on lesion dimensions, we compared 70 MRI examinations performed in 25 patients with definite MS, with the relevant clinical data as given by the Expanded Disability Status Scale (EDSS) and Functional System scale (FS). We found a significant correlation (r = 0.66, P = 0.0001) between the MRI score and the EDSS. Significant correlations also existed between MRI scores and cerebellar and brainstem FS scores. These correlations were consistently higher than those reported by other authors. We conclude that a standardized MRI examination, including sagittal protondensity and moderately T2-weighted images, should be performed in every MS patient. The improved clinical correlation could be of importance in follow-up studies when assessing the efficacity of therapy.

Localization of the mutation in an extended family with Charcot-Marie-Tooth neuropathy (HMSN I).

Hereditary motor and sensory neuropathy type I (HMSN I) or Charcot-Marie-Tooth (CMT) disease is an autosomal dominant peripheral neuropathy. In some CMT families linkage has been reported with either the Duffy blood group or the APOA2 gene, both located on chromosome 1q. More recently, linkage has been found in six CMT families with two chromosome 17p markers. We extensively analyzed a multi-generation Charcot-Marie-Tooth family by using molecular genetic techniques in order to localize the CMT gene defect. First, we constructed a continuous linkage group of 11 chromosome 1 markers and definitely excluded chromosome 1 as the site of mutation. Second, we analyzed the family for linkage with chromosome 17. The two-point lod scores obtained with D17S58 and D17S71 proved that this Charcot-Marie-Tooth family is linked to chromosome 17. Moreover, multipoint linkage results indicated that the mutation is most likely located on the chromosome 17p arm, distal of D17S71.

Lazarus sign and extensor posturing in a brain-dead patient. Case report.

A man was declared brain dead after having sustained a gunshot wound to the head. All clinical criteria for the diagnosis of brain death were met. The electroencephalogram was isoelectric, and four-vessel angiography demonstrated the absence of cerebral blood flow. However, stereotypic spontaneous movements were observed which persisted for several hours. The possible mechanism is discussed and a short review of the literature is given.