Experimental allergic encephalomyelitis in connexin 43-heterozygous mice.

Alterations in the expression of gap junction proteins (connexins) have previously been observed in experimental allergic encephalomyelitis (EAE). Demyelinating lesions have significantly decreased Cx43, while recovering lesions have greatly increased Cx43 and increased glial fibrillary acidic protein-expressing astrocytes. This suggests an important role for gap-junctional intercellular communication in astrocytes in the recovery from CNS inflammation. To study the effects of decreased Cx43 expression during acute disease (21 days post-immunization) and in recovering spinal cord tissue (55 days post-immunization) we induced EAE in Cx43 heterozygous and wild-type mice. Mice showed signs of disease by day 10, and signs of recovery by day 25. There were no clinical or pathological differences between heterozygous and wild-type mice in the acute disease stage, except that wild-type male mice had fewer clinical signs of disease. Male mice that were heterozygous for Cx43, and therefore had decreased expression of Cx43, had increased EAE disease severity. All demyelinating lesions had reduced numbers of reactive astrocytes and a significant decrease in Cx43 expression. In the 55-day study, all heterozygous and wild-type mice were clinically improved, showed decreased pathological signs, and showed increased laminin expression, indicative of CNS recovery. Furthermore, all heterozygous mice showed a striking increase in Cx43 expression during recovery, suggesting that the regulatory factors affecting Cx43 expression are still present in mice that have only one wild-type Cx43 allele.

Spontaneous remyelination following prolonged inhibition of alpha4 integrin in chronic EAE.

Inhibition of alpha(4)beta(1) integrin blocks immune cell influx into the CNS providing benefit to patients with multiple sclerosis and in animal model systems. We have used this mechanism to examine whether the presence of inflammatory cells suppresses spontaneous myelin repair in experimental autoimmune encephalomyelitis. We observed (1) 87% of plaques showed remyelination after 40 days of treatment; (2) myelin repair occurred in half of the total lesion area; (3) half of the animals regained motor function. There was no significant repair or gain of motor function in vehicle-treated animals. Therefore, prolonged inhibition of CNS inflammation, in the absence of targeted myelin repair, facilitates mechanisms of spontaneous remyelination.

Resolution-dependent estimates of multiple sclerosis lesion loads.

BACKGROUND: Changes in brain lesion loads assessed with magnetic resonance imaging obtained at 1.5 Telsa (T) are used as a measure of disease evolution in natural history studies and treatment trials of multiple sclerosis. METHODS: A comparison was made between the total lesion volume and individual lesions observed on 1.5 T images and on high-resolution 4 T images. Lesions were quantified using a computer-assisted segmentation tool. RESULTS: There was a 46% increase in the total number of lesions detected with 4 T versus 1.5 T imaging (p < 0.005). The 4 T also showed a 60% increase in total lesion volume when compared with the 1.5 T (p < 0.005). In several instances, the 1.5 T scans showed individual lesions that coalesced into larger areas of abnormality in the 4 T scans. The relationship between individual lesion volumes was linear (slope 1.231) showing that the lesion volume observed at 4 T increased with the size of the lesion detected at 1.5 T. The 4 T voxels were less than one quarter the size of those used at 1.5 T and there were no consistent differences between their signal-to-noise ratios. CONCLUSIONS: The increase in signal strength that accompanied the increase in field strength compensated for the loss in signal amplitude produced by the use of smaller voxels. This enabled the acquisition of images with improved resolution, resulting in increased lesion detection at 4 T and larger lesion volumes.

Apoptotic cells are present in the CNS throughout acute and chronic-progressive EAE in the absence of clinical recovery.

Experimental allergic encephalomyelitis (EAE) is an autoimmune, demyelinating disorder of the central nervous system induced in susceptible animals as a model for the human disease multiple sclerosis. Antibodies against the leukocyte adhesion molecule alpha4 integrin have been shown to prevent and reverse acute and chronic EAE of the guinea pig. The results presented in this paper implicate apoptosis as the mechanism of reversal of EAE following treatment with anti-alpha4 integrin antibody. Apoptotic cells were observed in the central nervous system (CNS) throughout chronic-progressive EAE of the guinea pig in the absence of clinical recovery. Many of the apoptotic cells were identified as T cells using immunohistochemistry. Similarly, apoptotic cells were present in the CNS of animals during anti-alpha4 integrin-mediated recovery from acute and chronic disease. Therefore, anti-alpha4 integrin-mediated recovery from EAE is due to the prevention of the influx of new inflammatory cells into the CNS that are required to replace those undergoing apoptosis.

Frontal, temporal, and striatal proton relaxation times in schizophrenic patients and normal comparison subjects.

Spin-lattice (T1) and spin-spin (T2) magnetic resonance relaxation times were examined in frontal, temporal, and striatal regions of 24 patients with schizophrenia and 10 normal comparison subjects. The schizophrenic patients had more prolonged T2 values than did the comparison subjects, particularly in the left temporal cortex and white matter, suggesting tissue pathology.

NMR studies in experimental allergic encephalomyelitis (EAE): normalization of T1 and T2 with parenchymal cellular infiltration.

The central nervous system (CNS) of guinea pigs undergoing acute experimental allergic encephalomyelitis (EAE) induced by whole CNS shows a characteristic progression of proton NMR relaxation times that correlates with pathologic features of the disease. Specifically, T1 was prolonged during meningeal infiltration and perivascular inflammation. T2 was increased with demyelination. Both proton T1 and T2 values, however, were normalized in active lesions containing an extensive cellular response (encephalitis, myelitis).

An open trial of OKT3 in patients with multiple sclerosis.

We report our experience with treatment with Muromonab-CD3 (Orthoclone OKT3) of 16 patients with multiple sclerosis (MS) who were in a progressive phase of their disease (n = 13) or in an acute severe attack lasting longer than 1 month without recovery (n = 3). We induced acute severe T-cytopenia with OKT3. Fifteen patients completed treatment for 10 days. Side effects were common and severe and included hypotension, nausea and vomiting, diarrhea, fever, and myalgia. In two of two patients tested, there was a transient though major rise in the levels of interferon gamma and tumor necrosis factor in the first 12 hours of treatment. Nonetheless, we did not detect new clinical or MRI activity of MS during the period of treatment, although many patients deteriorated transiently in disability scores. At the conclusion of follow-up, only four patients had deteriorated by 1.0 or more points on the Expanded Disability Status Scale of Kurtzke (EDSS) (73% stabilization rate). Of those patients who deteriorated, two died of complications of MS (EDSS 10). Only two patients had clinical improvement at 1 year follow-up. The attendant toxicity of OKT3 makes it unlikely that it will play a major role in the treatment of MS.

Evidence for a prolonged role of alpha 4 integrin throughout active experimental allergic encephalomyelitis.

The leukocyte integrin receptor, alpha 4 beta 1, and its endothelial cell ligand, vascular cell adhesion molecule 1, appear to be of critical importance in the leukocyte trafficking that accompanies CNS damage in experimental allergic encephalomyelitis (EAE). In this study, the persistence of the role for alpha 4 beta 1/VCAM-1 in EAE was established by observing antibody-mediated disease reversal up to 1 month following disease onset. Limited treatment with a monoclonal antibody against alpha 4 integrin, GG5/3, resulted in a significant decrease in both clinical and histopathologic signs. This was not observed in isotype control experiments. In the latter phase of progressive disease, widespread demyelination occurred in the animals that did not respond to 6 days of anti-alpha 4 treatment. These results demonstrate an essential role for alpha 4 beta 1 interactions throughout active EAE and illustrate the difference between reversible clinical deficits caused by edema and irreversible deficits associated with demyelination.

An open-trial evaluation of mitoxantrone in the treatment of progressive MS.

We treated 13 patients with progressive MS with mitoxantrone. All patients received a standard IV dose of mitoxantrone (8 mg/m2) every 3 weeks for a total of seven infusions, with dosage adjustments depending on the hematologic profile at the nadir. The treatment was well tolerated, with the most common side effect being mild nausea. Four of seven women developed transient secondary amenorrhea. The postenrollment clinical behavior of these patients was generally more favorable than during the 18 months prior to enrollment (only three of 13 patients developed an increase in the Expanded Disability Status Scale of more than 0.5 points), suggesting a possible treatment effect, but comparison with two historical control groups (both the active and placebo groups from the Canadian Cooperative Trial of Cyclophosphamide and Plasma Exchange) does not suggest that mitoxantrone was efficacious. Eight of 12 patients had evidence of MRI activity on 13 of 29 follow-up visits. This small, open-labeled pilot study did not provide strong support for proceeding with a randomized, controlled trial of this dosage regimen of mitoxantrone in patients with progressive MS.

A monoclonal antibody to alpha 4 integrin suppresses and reverses active experimental allergic encephalomyelitis.

In experimental allergic encephalomyelitis (EAE), circulating leukocytes enter the central nervous system (CNS) producing inflammation, myelin damage and paralysis. Prevention of leukocyte infiltration by an antibody against alpha 4 integrin suppressed clinical and pathological features of EAE in the guinea pig. Rapid clearance of leukocytes from the CNS and reversal of clinical findings were observed when anti-alpha 4 treatment was administered during active disease. Clinical improvement was accompanied by a marked decrease in abnormal pathological findings, including demyelination. Therefore anti-alpha 4 is an effective treatment of EAE and may be similarly useful in the treatment of autoimmune diseases such as multiple sclerosis.

Prolonged reversal of chronic experimental allergic encephalomyelitis using a small molecule inhibitor of alpha4 integrin.

CNS leukocytic invasion in experimental allergic encephalomyelitis (EAE) depends on alpha4beta1 integrin/vascular cell adhesion molecule-1 (VCAM-1) interactions. A small molecule inhibitor of alpha4beta1 integrin (CT301) was administered to guinea pigs in the chronic phase (>d40) of EAE for 10, 20, 30 or 40 days. CT301 elicited a rapid, significant improvement in the clinical and pathological scores that was maintained throughout the treatment period. A progressive loss of cells in the spinal cord of treated animals confirmed the resolution of inflammation associated with clinical recovery. Therefore, prolonged inhibition of alpha4beta1 integrin caused a sustained reversal of disease pathology in chronic EAE and may be similarly useful in MS.

Stability studies on chemoembolization mixtures. Dialysis studies of doxorubicin and lipiodol with Avitene, Gelfoam, and Angiostat.

RATIONALE AND OBJECTIVES: Chemoembolization, using a combination of embolic and chemotherapeutic agents, appears to be an effective treatment for hepatocellular carcinoma. Although the postulated mechanism of effectiveness hinges on a prolonged drug delivery, increasing evidence suggests that embolization mixtures are not stable. The objective of this study was to investigate examples of these mixtures. METHODS: Dialysis techniques have been used to examine the pharmacokinetic properties of chemoembolization mixtures that contain doxorubicin, Lipiodol (Guerbet Products, Montreal, Quebec), and the embolizing agents Avitene (Alcon Laboratories Inc., Fort Worth, Texas), Gelfoam (Upjohn, Kalamazoo, MI), and Angiostat (Regional Therapeutic Inc., Pacific Palisades, CA). RESULTS: Lipiodol, Gelfoam, and Avitene, when combined with doxorubicin, had only a small effect on the diffusion of the drug when compared with the diffusion curve of doxorubicin alone. Gelfoam or Avitene produced a thrombus-like consistency when added to a doxorubicin/Lipiodol combination, and an additional decrease in the doxorubicin appearance rate was observed. However, after 6 hours, doxorubicin levels for these mixtures reached control values observed in 3 hours. Angiostat without Lipiodol produced a profound concentration-dependent decrease in the diffusion of doxorubicin. After 9 hours, only 23% of the doxorubicin had been released. CONCLUSION: The strong complexing ability of the embolic agent Angiostat may enable it to be a carrier for doxorubicin and surpass other mixtures currently employed for transcatheter chemoembolization.

Magnetic resonance imaging of small medullary infarctions.

Small infarctions of the medulla produce typical neurologic deficits and can be clinically recognized fairly reliably. High-field magnetic resonance imaging with a 1.5 T prototype scanner successfully demonstrated very tiny medulla infarctions in three of five patients. These lesions were imaged readily by a fairly rapid (approximately 1-2 min scan time) partial-saturation technique and confirmed on multiple spin-echo images. In addition, in two cases vertebral occlusion ipsilateral to the infarction was suggested by high signal on T2 weighted spin-echo images.

Quantification of multiple sclerosis lesion volumes in 1.5 and 0.5 T anisotropically filtered and unfiltered MR exams.

Recently, guidelines for the use of MRI in the monitoring of MS have recommended the use of imaging systems with mid-field (0.5-1.0 T) or high-field (greater than 1.0 T) strengths. Higher field strengths provide many advantages, including increased signal-to-noise ratios (SNR). SNR also may be increased by post-processing algorithms that reduce noise. In this paper we evaluate the impact on operator variability of (a) lesion quantification in high-field (1.5 T) versus mid-field (0.5 T) exams; and (b) an anisotropic diffusion filter algorithm that reduces image noise without blurring or moving object boundaries. Inter- and intra-operator reliability and variability were studied using repeated quantification of lesions in 1.5 and 0.5 T filtered and unfiltered MR exams of a MS patient. Results indicate that inter-operator variability in 1.5 T unfiltered exams was 0.34 cm3 and was significantly larger than that in 1.5 T filtered (0.27 cm3), 0.5 T unfiltered (0.26 cm3), and 0.5 T filtered (0.24 cm3) exams. Similarly, intra-operator variability in 1.5 T unfiltered exams was 0.23 cm3 and was significantly larger than that in 1.5 T filtered (0.19 cm3), 0.5 T unfiltered (0.19 cm3), and 0.5 T filtered (0.18 cm3) exams. In addition, the minimum significant change between two successive measurements of lesion volume by the same operator, was 0.64 cm3 in 1.5 T unfiltered exams, but 0.53 cm3 or less in other exams. For two different operators making successive measurements, the minimum significant change was 0.94 cm3 in 1.5 T unfiltered exams, but only 0.75 cm3 or less in other exams. Finally, the number of lesions to be monitored for an average change in volume at a given power and significance level was greater by 30%-60% for quantification in 1.5 T unfiltered exams. These results suggest that inter- and intra-operator variability are reduced by anisotropic filtering, and by quantification in 0.5 T exams. Reduced operator variabilities may result from higher detail signal-to-noise ratios (dSNRs) in 0.5 T and filtered exams.

The variability of manual and computer assisted quantification of multiple sclerosis lesion volumes.

The high resolution and excellent soft tissue contrast of Magnetic Resonance Imaging (MRI) have enabled direct, noninvasive visualization of Multiple Sclerosis (MS) lesions in vivo. This has allowed the quantification of changes in the appearance of lesions in MR exams to be used as a measure of disease state. Nevertheless, accurate quantification techniques are subject to inter- and intra-operator variability, which may hinder monitoring of disease progression. We have developed a computer program to assist an experienced operator in the quantification of MS lesions in standard spin-echo MR exams. The accuracy of assisted and manual quantification under known conditions was studied using exams of a test phantom, while inter- and intra-operator reliability and variability were studied using exams of a MS patient. Results from the phantom study show that accuracy is improved by assisted quantification. The patient exam results indicate that assisted quantification reduced inter-operator variability from 0.34 to 0.17 cm3, and reduced intra-operator variability from 0.23 to 0.15 cm3. In addition, the minimum significant change between two successive measurements of lesion volume by the same operator was 0.64 cm3 for manual quantification and 0.42 cm3 for assisted quantification. For two different operators making successive measurements, the minimum significant change was 0.94 cm3 for manual quantification, but only 0.47 cm3 for assisted quantification. Finally, the number of lesions to be monitored for an average change in volume at a given power and significance level was reduced by a factor of 2-4 by assisted quantification. These results suggest that assisted quantification may have practical applications in clinical trials, especially those that are large, multicenter, or extended over time, and therefore require lesion measurements by one or more operators.

Brain retractor edema during induced hypotension: the effect of the rate of return of blood pressure.

This study evaluated the hypothesis that the postoperative formation of cerebral edema may be influenced by the rate of blood pressure return after induced hypotension in a graded brain retractor injury. Nineteen cats underwent unilateral craniotomy, isoflurane-induced hypotension to a mean of 50 mm Hg, and application of a brain retractor at 20 mm Hg of pressure for 1 hour. Blood pressure was returned to normal either within 3 minutes or over 20 minutes. The degree of cerebral edema formation was determined by Evans blue dye and coronal magnetic resonance imaging. All animals showed extravasation of Evans blue dye in the retracted hemisphere that was most marked at the periphery of the retractor. T1 relaxation times were significantly prolonged in the retracted hemispheres of both the fast return and slow return groups (18.8% and 17.8%, respectively) and more so at the Evans blue sites (42.8% and 40.8%), although not so strikingly beneath the retractor itself (6.3% and 7.8%). T2 relaxation times were similarly prolonged but to approximately half the degree of the T1 times. In the nonretracted hemisphere, drug-induced hypotension alone did not result in significant acute cerebral edema or blood-brain barrier alteration. There was no significant difference between the fast and slow groups in Evans blue extravasation or magnetic resonance changes. Thus, in a retractor-induced brain injury, restoration of arterial pressure to normal either gradually or rapidly did not influence the degree or extent of edema formation.

Reversible toroidal compaction of DNA by aluminum.

Electron microscopy has been used to characterize the products of the reactions of aluminum with DNA under three different conditions, one of which, pH 5 and Al (III)/DNA(P) ratio of 0.4, has been previously shown to produce reversible interstrand crosslinking in double-stranded DNA molecules. Under this condition, aluminum produced macromolecular aggregates of DNA upon heating, with a distinctive ultrastructure reversible to double-stranded DNA after removal of the aluminum. These structures were toroidal in configuration and exhibited mean widths of 4.9 +/- 1.8 nm and were 18.6 +/- .4 nm in diameter with a toroidal internal diameter of 7.4 +/- 4.7 nm. Previous results have shown that these structures contain Al(III) - crosslinked DNA, the present data suggests that this intermolecular crosslinking is associated with the production of compacted structures.

Polynucleotide cross-linking by aluminum.

The observations that there was an increased concentration of Al in the brains of Alzheimer's, Guam-Parkinson, and amyotrophic lateral sclerosis disease patients and that there was an apparent localization of the Al in chromatin led to a study of the interaction of Al(III) with DNA. We have previously shown that Al cross-links calf thymus DNA at low pH (S. J. Karlik, G. L. Eichhorn, P. N. Lewis, and D. R. Crapper, Biochemistry 19, 5991 [1980]). Extended studies indicate that cross-linking occurs in DNAs of all base ratios, including polydAdT and polydGdC. Since Al cross-links prevent renaturation in polydAdT, the decrease in the amount of polymer renatured in the presence of Al becomes a quantitative appraisal of the extent of cross-linking. Saturation of cross-linking occurs at a 0.4 ratio of Al to nucleotide phosphate, indicating that potentially 80% of the base pairs are Al bound. Cross-links are broken at elevated pH and by EDTA.

NMR studies in the relapsing experimental allergic encephalomyelitis (EAE) model of multiple sclerosis in the strain 13 guinea pig.

Juvenile strain 13 guinea pigs sensitized with an emulsion of whole isologous central nervous system (CNS) tissue in complete Freund's adjuvant in the first two weeks of life develop a relapsing-remitting form of experimental allergic encephalomyelitis (EAE) which resembles multiple sclerosis (MS) both clinically and pathologically. In order to determine if this experimental model could be used to identify the tissue factors which contribute to the magnetic resonance imaging (MRI)-detected lesions in MS, we measured T1 and T2 relaxation times, tissue specific gravity and histology throughout the entire CNS in vitro in eighteen animals during the acute phase (first attack), and twenty-one animals during further periods of clinical worsening (relapses) and recovery (remissions). The neuropathological findings of spinal cord meningeal inflammation, perivascular and parenchymal infiltration (myelitis and encephalitis) and demyelination were more marked during periods of clinical worsening than when the animal had recovered clinically. Even though the histological changes of EAE were present in all experimental animals, NMR relaxation times and tissue specific gravity could not distinguish experimental (first attack, subsequent relapses and remissions) from control animals due to a wide range of values for each of these parameters. Although relapsing EAE in the strain 13 guinea pig has been an instructive model of MS, we have found that in vitro NMR relaxometry cannot be used to predict the presence or degree of pathological change in the nervous system of these experimental animals.

Correlation between MRI and clinico-pathological manifestations in Lewis rats protected from experimental allergic encephalomyelitis by acylated synthetic peptide of myelin basic protein.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system which constitutes an accepted animal model for multiple sclerosis (MS). The disease can take an acute or chronic form depending on the injection route, animal strain and nature of the disease-inducing antigen administered. The neuroinflammation associated with the acute form can be detected with T2-weighted, T1-weighted and diffusion MRI, and blood-brain barrier changes can be investigated with Gd-DTPA-enhanced T1-weighted imaging, similar to that of MS patients. A synthetic peptide of myelin basic protein (MBP) encephalitogenic for the Lewis rat (MBP 68-86) was acylated by the attachment of a palmitoyl residue (PAL68-86), and was shown to confer almost complete protection against EAE, when administered to rats before and after an encephalitogenic challenge. In this study, treatment of Lewis rats with PAL68-86 prevented the appearance of clinical signs (p < 0.0001) after challenge with the native peptide (p68-86) in complete Freund's adjuvant (CFA), and reduced considerably the MRI and histopathological signs of the disease (p < 0.0001). Measurement of the gadolinium leakage due to neuroinflammation revealed a significant decrease in permeability from 4.09 +/- 2.1 to 2.95 +/- 1.79% pixels > mean + 2 SD (p = 0.011). Therefore, quantitative MRI measurements correlate very well with the reduced cellular infiltration in the CNS and the absence of clinical signs in the EAE-protected animal.