Mitochondrial myopathy: correlation between oxidative defect and mitochondrial DNA deletions at single fiber level

In situ hybridization combined with immunohistochemical techniques has been applied to study patients affected by mitochondrial myopathies with large mitochondrial (mt)DNA deletions. All patients' muscle biopsies showed ragged red fibers (RRFs) and cytochrome oxidase (COX) deficiency. Two digoxygenin-labeled, polymerase chain reaction (PCR)-amplifed DNAs were used as probes. One probe was designed to hybridize only with wild-type mtDNAs, while the other recognized both wild-type and deleted mtDNAs. Concomitant immunocytochemical analysis using antibodies against subunits II, III, (encoded by mtDNA) and IV (encoded by nuclear DNA) of COX was carried out. In our patients deleted mtDNAs are overexpressed in COX-negative RRFs, while wild-type mtDNAs are decreased in the same fibers. Immunohistochemistry studies show that COX IV is overexpressed in RRFs and that COX II and COX III subunits are still present. Deleted mtDNAs are spatially segregated in muscle fibers, where they interfere with the local population of normal mitochondrial genomes, causing a regional deficiency of the mitochondrial respiratory activity.This work was supported by the Associazione Amici del Centro Dino Ferrari     

Magnetization-transfer histogram analysis of the cervical cord in patients with multiple sclerosis

BACKGROUND AND PURPOSE: Previous studies have failed to show significant correlations between the number and extent of T2 spinal cord lesions and the clinical status of multiple sclerosis (MS) patients. We evaluated 1) whether it is feasible to create magnetization transfer-ratio (MTR) histograms of the cervical cord in patients with MS by using two different acquisition schemes, and 2) whether cervical cord MTR histogram metrics were different from those of healthy control subjects and between MS patients with and without locomotor disability. METHODS: We obtained two sets of gradient-echo sequences with and without a saturation pulse from 90 MS patients and 20 sex- and age-matched healthy control subjects. One set consisted of 20 axial, contiguous slices with a thickness equal to 5 mm. The other set consisted of 17 sagittal slices with a thickness equal to 3 mm and an interslice gap equal to 0.3 mm. After image coregistration and removal of tissues around the cervical cord, MTR histograms were created. The average MTR, the peak height, and the peak position of the histograms were measured. All of these measurements were from the whole of the cervical cord, thus including both MS lesions and normal-appearing tissue. RESULTS: When comparing the MTR histograms obtained using axial, contiguous, 5-mm-thick slices, MS patients had significantly lower average cervical cord MTR and peak height than did control subjects. When comparing the MTR histograms obtained using sagittal, 3-mm-thick slices, MS patients also had significantly lower average cervical cord MTR and peak location than did control subjects. Patients with locomotor disability had significantly lower average cord MTR and peak location than those without. CONCLUSION: This study shows that it is feasible to obtain MTR histograms of the cervical cord from MS patients by using different acquisition schemes. Our results also suggest that the assessment of MS cervical cord damage, achieved using MTR histograms, may lead to a better understanding of the clinical manifestations of the disease.     

A comparison of MR imaging with fast-FLAIR, HASTE-FLAIR, and EPI-FLAIR sequences in the assessment of patients with multiple sclerosis

BACKGROUND AND PURPOSE: Fast fluid-attenuated inversion-recovery (FLAIR) sequences are sensitive for detecting lesions in patients with multiple sclerosis (MS). More rapid fast-FLAIR imaging of the brain can be achieved by the concomitant use of half-Fourier acquisition single-shot turbo spin-echo (HASTE-FLAIR) and echo-planar imaging (EPI-FLAIR). The present study was performed in a large cohort of subjects to assess and compare the number and volume of brain lesions detected by the fast-FLAIR, HASTE-FLAIR, and EPI-FLAIR sequences in patients with MS. METHODS: Fast-FLAIR, HASTE-FLAIR, and EPI-FLAIR sequences were obtained from 46 consecutive MS patients. Lesions seen on each type of sequence were counted and classified by consensus by two observers. Lesion volumes were measured using a semiautomated segmentation technique based on local thresholding. RESULTS: The quality of the fast-FLAIR images was significantly better than that of HASTE-FLAIR and EPI-FLAIR images. Fast-FLAIR revealed significantly more lesions and higher lesion volumes than did HASTE-FLAIR and EPI-FLAIR. A similar number of large lesions was detected by the three sequences, but HASTE-FLAIR and EPI-FLAIR showed significantly fewer small and intermediate lesions than did fast-FLAIR. The number of lesions seen on HASTE-FLAIR and EPI-FLAIR images was similar. CONCLUSION: HASTE-FLAIR and EPI-FLAIR sequences revealed as many large MS lesions as fast-FLAIR. Because their acquisition times are only a fraction of that needed for fast-FLAIR sequences, they may be useful for making a rapid diagnosis of MS in uncooperative patients. Their reduced ability to detect smaller lesions indicates that they should not be used as a routine approach to imaging patients with MS.     

Neurophysiological investigations in multiple sclerosis

The advent of magnetic resonance imaging techniques has greatly reduced the diagnostic value of neurophysiological tests, particularly evoked potentials, in multiple sclerosis patients, because of the higher sensitivity in revealing subclinical involvement of the central nervous system. Technical progress and new methods of investigating afferent and efferent nervous pathways would seem to increase the sensitivity in detecting neural dysfunction, but the 'clinical gain' is modest at best. More promising is the utilization of neurophysiological tests to quantify the severity of white matter involvement. Transversal and longitudinal studies have demonstrated good correlations between neurophysiological parameters and disability measures, indicating that a battery of neurophysiological tests could be useful in monitoring the disease evolution in single patients and as surrogate endpoints in clinical trials. Further studies are needed for a better definition of the applications of evoked potentials and other neurophysiological techniques. Finally, event-related potentials and advanced electroencephalogram techniques, such as coherence analysis, could provide useful information on the pathophysiology of cognitive dysfunction, so common in multiple sclerosis patients, and with a strong impact on the quality of life.     

Bacterial and mammalian DNA alkyltransferases sensitize Escherichia coli to the lethal and mutagenic effects of dibromoalkanes

Here we confirm and extend our previous studies demonstrating that the mutagenic potency of 1,2-dibromoethane (DBE) and dibromomethane (DBM) is markedly enhanced (not prevented) in bacteria expressing the O6- alkylguanine-DNA alkyltransferase (ATase) encoded by the Escherichia coli ogt gene. We demonstrate that, in close parallel with mutagenesis, the Ogt ATase sensitizes the bacteria to the lethal effects of these carcinogens, suggesting that one or more of the potentially mutagenic lesions induced by DBE and DBM in the presence of Ogt has additional lethal capacity. We further demonstrate that the sensitization to both lethality and mutagenesis by DBE and DBM is a property shared by other DNA alkyltransferases. This objective was accomplished by quantifying the induction of mutations and lethal events in ogt- ada- E. coli expressing an exogenous bacterial or mammalian ATase from a multicopy plasmid. Mammalian recombinant ATases enhanced the lethal and mutagenic actions of DBE and suppressed the lack of sensitivity of the vector- transformed bacteria to DBM. In most cases the order of effectiveness of the ATases ranked: murine > human > Ogt > rat. Further comparisons included the full-length Ada ATase from E. coli and a truncated Ada version (T-ada) that retains the O6-methylguanine binding domain of the protein. The full-length Ada ATase was effective in enhancing the lethality but not the mutagenicity induced by DBE and DBM. The T-ada ATase provided less sensitization than Ada to lethality by DBE, but of the three bacterial ATases T-ada yielded the highest sensitization to mutagenesis by this compound. T-ada and Ada ATases were in general less effective than the mammalian versions, with the exception of the rat recombinant ATase. The effectiveness of the different mammalian and bacterial ATases in promoting the deleterious actions of dibromoalkanes was compared with the effectiveness of these proteins in suppressing the lethal and mutagenic effects induced by N-nitroso-N-methylurea. The ability to sensitize E. coli to the lethal and mutagenic effects of DBE and DBM seems restricted to DNA alkyltransferase, since overexpression of thioredoxin (Trx) or glutaredoxin (Grx1) in ogt- ada- cells showed no effect, in spite of the reported potential of bioactive dihaloethane- derived species to alkylate Trx.      

Guidelines for autologous blood and marrow stem cell transplantation in multiple sclerosis: a consensus report written on behalf of the European Group for Blood and Marrow Transplantation and the European Charcot Foundation

Recent reports suggest the possible beneficial effects of haemopoietic stem cell transplantation (HSCT) in autoimmune diseases such as multiple sclerosis (MS). The definition of the risk/benefit ratio for such a treatment is perceived as a major issue for the neurological community worldwide. The First Consensus Conference on Bone Marrow Transplantation in Patients with Multiple Sclerosis was held in Milan, Italy on 21 February 1998. Participants from 16 European, North American, and South American countries discussed the guidelines form performing HSCT in MS. This conference was organized in order to : (a) define criteria for patient selection; (b) define transplantation procedures to maximize efficacy of the treatment and minimize its toxicity; (c) standardize patient outcome evaluation; and (d) establish an international working group to evaluate the efficacy and safety of HSCT in MS and to study the immunological changes related to HSCT in MS patients. During the meeting in Milan agreement was reached on: (a) the preparation and distribution of a consensus report on HSCT in MS and (b) the design of an open trial for an initial assessment of the safety and efficacy of HSCT in MS. The consensus reached during the meeting and the design of the clinical trial are summarized in this contribution. Received: 14 May 1999, Received in revised form: 4 January 2000, Accepted: 19 January 2000     

Neurocognitive profiles in Duchenne muscular dystrophy and gene mutation site

The presence of nonprogressive cognitive impairment is recognized as a common feature in a substantial proportion of patients with Duchenne muscular dystrophy. To investigate the possible role of mutations along the dystrophin gene affecting different brain dystrophin isoforms and specific cognitive profiles, 42 school-age children affected with Duchenne muscular dystrophy, subdivided according to sites of mutations along the dystrophin gene, underwent a battery of tests tapping a wide range of intellectual, linguistic, and neuropsychologic functions. Full-scale intelligence quotient was approximately 1 S.D. below the population average in the whole group of dystrophic children. Patients with Duchenne muscular dystrophy and mutations located in the distal portion of the dystrophin gene (involving the 140-kDa brain protein isoform, called Dp140) were generally more severely affected and expressed different patterns of strengths and impairments, compared with patients with Duchenne muscular dystrophy and mutations located in the proximal portion of the dystrophin gene (not involving Dp140). Patients with Duchenne muscular dystrophy and distal mutations demonstrated specific impairments in visuospatial functions and visual memory (which seemed intact in proximally mutated patients) and greater impairment in syntactic processing.     

Signal transduction by the platelet Fc receptor

We have evaluated the mechanism by which crosslinking human platelet Fc receptor (FcR) for IgG triggers platelet aggregation and the platelet release reaction. Platelet FcR was crosslinked by incubating purified human platelets with anti-FcRII monoclonal antibody and F(ab')2 anti- mouse Ig. The resultant [Ca2+]i increase, monitored by Fura-2 and measured in the absence of extracellular Ca2+, reached a peak of 750 +/- 50 nmol/L. The effects of cyclooxygenase inhibitors, aspirin and indomethacin, and a phospholipase A2 inhibitor, dibromoacetophenone, were examined. Regardless of the inhibitor, at least 25% of the [Ca2+]i increase remained. Thrombin (0.2 U/mL) stimulated an immediate [Ca2+]i increase that reached 1.95 +/- 0.8 mumol/L. The [Ca2+]i increase generated by thrombin was only slightly reduced by these inhibitors. Crosslinking the FcRII of platelets resulted in a fivefold increase in the production of [3H]inositol phosphates, (IP) which, in the absence of extracellular Ca2+ was insensitive to aspirin. The activation of a [Ca2+]i increase along with the measured increases in IP indicate that FcRII crosslinking leads to the activation of phospholipase C (PLC). In contrast to thrombin, platelet activation via FcRII depends to a large extent on arachidonic acid metabolites. However, neither cyclooxygenase nor phospholipase A2 inhibitors completely blocked FcRII-stimulated [Ca2+]i increase. These observations led us to propose that crosslinking of platelet FcRII initially activates PLC.