Stratification and monitoring of natalizumab-associated progressive multifocal leukoencephalopathy risk: recommendations from an expert group.

The use of natalizumab for highly active relapsing-remitting multiple sclerosis (MS) is influenced by the occurrence of progressive multifocal leukoencephalopathy (PML). Through measurement of the anti-JCV antibody index, and in combination with the presence or absence of other known risk factors, it may be possible to stratify patients with MS according to their risk of developing PML during treatment with natalizumab and detect early suspected PML using MRI including a diffusion-weighted imaging sequence. This paper describes a practical consensus guideline for treating neurologists, based on current evidence, for the introduction into routine clinical practice of anti-JCV antibody index testing of immunosuppressant-naïve patients with MS, either currently being treated with, or initiating, natalizumab, based on their anti-JCV antibody status. Recommendations for the frequency and type of MRI screening in patients with varying index-associated PML risks are also discussed. This consensus paper presents a simple and pragmatic algorithm to support the introduction of anti-JCV antibody index testing and MRI monitoring into standard PML safety protocols, in order to allow some JCV positive patients who wish to begin or continue natalizumab treatment to be managed with a more individualised analysis of their PML risk.

c-Jun N-terminal kinase inhibitor SP600125 modulates the period of mammalian circadian rhythms.

Circadian rhythms are endogenous cycles with periods close to, but not exactly equal to, 24 h. In mammals, circadian rhythms are generated in the suprachiasmatic nucleus (SCN) of the hypothalamus as well as several peripheral cell types, such as fibroblasts. Protein kinases are key regulators of the circadian molecular machinery. We investigated the role of the c-Jun N-terminal kinases (JNK), which belong to the mitogen-activated protein kinases family, in the regulation of circadian rhythms. In rat-1 fibroblasts, the p46 kDa, but not the p54 kDa, isoforms of JNK expressed circadian rhythms in phosphorylation. The JNK-inhibitor SP600125 dose-dependently extended the period of Period1-luciferase rhythms in rat-1 fibroblasts from 24.23+/-0.17-31.48+/-0.07 h. This treatment also dose-dependently delayed the onset of the bioluminescence rhythms. The effects of SP600125 on explant cultures from Period1-luciferase transgenic mice and Period2(Luciferase) knockin mice appeared tissue-specific. SP600125 lengthened the period in SCN, pineal gland, and lung explants in Period1-luciferase and Period2(Luciferase) mice. However, in the kidneys circadian rhythms were abolished in Period1-luciferase, while circadian rhythms were not affected by SP600125 treatment in Period2(Luciferase) mice. Valproic acid, already known to affect period length, enhanced JNK phosphorylation and, as predicted, shortened the period of the Period1-bioluminescence rhythms in rat-1 fibroblasts. In conclusion, our results showed that SP600125 treatment, as well as valproic acid, alters JNK phosphorylation levels, and modulates the period length in various tissues. We conclude that JNK phosphorylation levels may help to set the period length of mammalian circadian rhythms.

Quantitative MRI in patients with secondary progressive MS treated with monoclonal antibody Campath 1H.

BACKGROUND: To assess the long-term effect of the lymphocyte-depleting humanized monoclonal antibody Campath 1H on MR markers of disease activity and progression in secondary progressive MS patients. METHODS: Twenty-five patients participated in a crossover treatment trial with monthly run-in MR scans for 3 months, followed (after a single pulse of Campath 1H) by monthly MR scans from months 1 to 6 and again from months 12 to 18. MR analysis was performed to provide measurements of the number and volume of gadolinium (Gd)-enhancing lesions as well as the hypointense lesion volume on a T1-weighted sequence. In addition, serial measurements of T2 brain lesion volume, brain volume, and spinal cord cross-sectional area were made over the duration of the study. The relationship between clinical and MR measures of disease evolution was also assessed. RESULTS: Treatment was associated with a reduction in the number and volume of Gd-enhancing lesions (p < 0.01). Despite this, a decrease in brain volume was seen in 13 patients during the 18 months post-treatment. The mean pretreatment Gd-enhancing lesion volume was predictive of subsequent reduction in brain volume (r = 0.77, p = 0.002). Reduction in brain volume also correlated with the change in T1 hypointense lesion volume after treatment (r = 0.53, p < 0.01). A reduction in spinal cord area was also seen throughout the study duration, and this correlated with an increase in disability (r = 0.65, p = 0.01). CONCLUSION: Campath 1H treatment was associated with a sustained and marked reduction in the volume of Gd enhancement, indicating suppression of active inflammation. Nevertheless, many patients developed increasing brain and spinal cord atrophy, T1 hypointensity, and disability. This study highlights the potential role for novel MR techniques in monitoring the effect of treatment on the pathologic process in MS.

Sample size estimations for MRI-monitored trials of MS comparing new vs standard treatments.

The authors estimated the sample sizes needed for exploratory trials of MS assessing the efficacy of new treatments in reducing the number of new enhancing lesions vs those of interferon-beta or glatiramer acetate. The sample sizes per arm ranged from 868 (effect: 20%) to 94 (effect: 50%) for patients with relapsing-remitting MS and from 2,484 (effect: 20%) to 361 (effect: 50%) for patients with secondary progressive MS. In MS, exploratory trials of new vs available therapies require large numbers of patients, even when MR end-points are used.

Improving interobserver variation in reporting gadolinium-enhanced MRI lesions in multiple sclerosis.

Gadolinium-enhanced MRI is a sensitive and objective means to monitor disease activity in multiple sclerosis (MS). We evaluated the interobserver agreement and the value of observer training in reporting enhancing lesions from serial MRI. Scans of 16 MS patients were evaluated by five inexperienced and five experienced observers before and after consensus formation and training. The number of lesions at baseline, and the number of new and persistent lesions at follow-up were scored. For each condition, weighted kappa values (kappa) and the mean average difference to the median (MADM) scores were calculated. Without training, the experienced readers showed good agreement on number of lesions at baseline and new lesions at follow-up, and moderate agreement for persistent lesions. The inexperienced readers showed poor agreement for baseline and persistent lesions, and moderate agreement for new lesions. After training, both groups reported lower absolute numbers of lesions, especially the inexperienced readers. The experienced readers showed good agreement for all lesion types, the inexperienced readers showed agreement for baseline and new lesions, and agreement was moderate for persistent lesions. In both groups MADM scores were < 0.72 for baseline and new lesions, but > 1.2 for persistent lesions. Interobserver agreement is improved by training, especially in inexperienced readers. Interobserver agreement in reporting gadolinium-enhanced lesions is high, which validates the use of serial, enhanced MRI as an outcome parameter in treatment trials in MS.

The effect of IFNbeta-1b on the evolution of enhancing lesions in secondary progressive MS.

BACKGROUND: After the resolution of contrast enhancement, the majority of new MS lesions become isointense with surrounding white matter on T1-weighted MRI. Less commonly, a hypointense T1 lesion develops, representing the development of more severe focal tissue damage. Interferon beta (IFNbeta) reduces both the number of new enhancing lesions and the duration of contrast enhancement. OBJECTIVE: To determine if IFNbeta affects the degree of tissue damage within new lesions and if its effects are related to lesion size. METHODS: One hundred twenty-five patients with secondary progressive MS from seven European sites were randomized to receive either IFNbeta-1b or placebo. Monthly, contrast-enhanced T1-weighted MR images were acquired at baseline, at months 1 to 6, and at months 19 to 24. The size of all new enhancing lesions developing between months 1 and 6 was recorded and their appearance at follow-up documented. RESULTS: In the first 6 months, fewer new enhancing lesions occurred in the IFNbeta-1b arm. This difference was greater for small (70% decrease) than for large (46% decrease) lesions. Hypointense T1 lesions were more likely to form from large (25%) than from small (9%) enhancing lesions in both treatment arms. Patients taking IFNbeta-1b developed fewer hypointense T1 lesions; however, the proportion of enhancing lesions developing into hypointense T1 lesions was similar in both arms. CONCLUSION: IFNbeta-1b reduced the number of new enhancing lesions, with a greater effect on small lesions. However, when a new enhancing lesion did become established, treatment with IFNbeta-1b did not alter its subsequent course.

Clinical-MRI correlations in a European trial of interferon beta-1b in secondary progressive MS.

BACKGROUND: The recently completed placebo-controlled multicenter randomized trial of interferon beta-1b (Betaferon) in 718 patients with secondary progressive MS shows significant delay of disease progression and reduction of relapse rate. This study provides an opportunity to assess the level of relationship between clinical and MRI outcomes in this cohort of patients with secondary progressive MS. METHODS: Brain T2-weighted lesion volume was measured annually in all available patients, with visual analysis to identify any new or enlarging (active) T2 lesions at each annual time point. A subgroup of 125 patients had monthly gadolinium-enhanced, T1-weighted imaging at months 0 to 6 and 18 to 24. Relapses were documented and expanded disability status scale (EDSS) was measured every 3 months. RESULTS: For the annual MRI outcomes, a significant but modest correlation was identified between the change in T2 lesion volume from baseline to the final scan and the corresponding change from baseline in EDSS (r = 0.17, p < 0.0001). There were significant correlations between the cumulative number of active T2 lesions and 1) change in EDSS (r = 0.18, p < 0.0001) and 2) relapse rate (r = 0.24, p < 0.0001). In the subgroup of 125 patients undergoing monthly imaging, MRI lesion activity was correlated with relapse rate over months 0 to 24 (r = 0.24, p = 0.006) but not with change in EDSS. CONCLUSIONS: These results confirm that the clinical-MRI relationships previously identified in relapsing-remitting MS still are apparent in the secondary progressive phase of the disease and support the use of MRI as a relevant outcome measure. In view of the relatively modest nature of the correlations, it seems unwise to rely on such MRI measures alone as primary efficacy variables in secondary progressive MS trials.

The sensitivity of thin-slice fast spin echo, fast FLAIR and gadolinium-enhanced T1-weighted MRI sequences in detecting new lesion activity in multiple sclerosis.

Fast fluid-attenuated inversion-recovery (FLAIR) and proton density/T2-weighted fast spin echo (FSE) brain images with 3-mm slices were acquired monthly for 7 months in 37 multiple sclerosis patients. New and enlarging lesions were counted and compared according to the site of lesions seen with each sequence. In addition, the number of new enhancing lesions seen on gadolinium-enhanced T1-weighted brain magnetic resonance imaging at the same time points was counted. All sequences used 3-mm contiguous axial slices. Overall, 126 new or enlarging lesions were seen on FSE and 135 on fast FLAIR (P = 0.25, Wilcoxon signed ranks test). Regional comparisons revealed significantly more fast FLAIR lesions only in the cortical/subcortical areas. There was a total of 295 new enhancing lesions over the same period -- a gain in the number of 'active lesions' of 234% seen with FSE and 218% with FLAIR. It is concluded that serial thin slice fast FLAIR is only slightly superior to FSE in detecting new and enlarging multiple sclerosis lesions but the difference is not sufficient to recommend that FLAIR should replace FSE in short-term, exploratory trials in MS using monthly scanning. Gadolinium-enhanced imaging is more then twice as sensitive as either FSE or fast FLAIR to new multiple sclerosis lesion activity, and enhancing lesions should provide the primary outcome measure in such studies.

The use of magnetic resonance imaging in multiple sclerosis treatment trials: power calculations for annual lesion load measurement.

Phase III definitive treatment trials of new multiple sclerosis (MS) therapies now routinely incorporate an annual magnetic resonance imaging protocol, with change in T2-weighted brain lesion load providing an important outcome measure. To date the accepted strategy has been to perform a core imaging protocol on all patients in such studies. The aim of this study was to provide power calculations based on this MRI endpoint. Serial MRI data from 128 patients with either relapsing remitting (RR) or secondary progressive (SP) MS were used to calculate sample size requirements using a repeated measures analysis of variance design. We provide sample size calculations based on various follow-up intervals and effect sizes. Sample sizes for the SPMS cohort were substantially larger than for the RRMS group, reflecting the greater variance in lesion load changes between patients in the SPMS group. With a follow-up of 3 years, we estimate that only 12 and 33 patients per arm are needed to show stabilisation of MRI lesion load in the RRMS and SPMS groups, respectively. Our results suggest that ongoing phase III treatment trials are more than adequately powered to detect even subtle treatment effects, and indicate that incorporating measurements from longer follow-up durations increases power substantially. We conclude that an annual imaging protocol provides a robust and powerful tool for assessing effects on the radiological appearance of the disease process.

The effect of section thickness on MR lesion detection and quantification in multiple sclerosis.

BACKGROUND AND PURPOSE: The purpose of our study was to investigate the effect of section thickness on MR detection of brain lesion volume and measurement precision in patients with multiple sclerosis (MS). METHODS: Eight subjects with known MS were studied on a 1.5-T MR system. We used a 3D fast fluid-attenuated inversion-recovery sequence to obtain contiguous axial brain images at section thicknesses of 5 mm, 3 mm, and 1 mm. Two sets of images were acquired at each section thickness during two sessions, between which the patient was removed from the scanner. Lesion volumes were measured at each section thickness using a semiautomated local thresholding technique. RESULTS: We found that progressive reduction in section thickness led to detection of smaller lesions, resulting in a significant (8%) increase in lesion volume on MR images as section thickness was reduced from 5 mm to 3 mm. However, despite a further increase in lesion detection at a section thickness of 1 mm, this did not result in an increase in total lesion volume. This finding indicates that the relationship between section thickness and lesion volume on MR images is not linear. Scan-rescan reproducibility was improved by reducing section thickness, at the cost of increased analysis time. CONCLUSION: This study shows that acquisition of very thin sections increases the sensitivity and precision of MS lesion measurement. Serial studies assessing lesion changes over time are needed to define the impact of this increase on sample size requirements for MS treatment trials.

Proton MR spectroscopy in clinically isolated syndromes suggestive of multiple sclerosis.

The concentration of the metabolite N-acetyl aspartate (NAA), thought to be a marker of axonal loss or damage, has been shown to be reduced in lesions, as demonstrated by high signal areas on T2-weighted MRI, and in normal-appearing white matter (NAWM) in established multiple sclerosis (MS). The stage of the disease when these changes first appear is not known. To try to determine this we studied 20 patients with clinically isolated syndromes, many of whom will be at the earliest clinical stages of MS, and 20 age- and sex-matched controls with single-voxel proton magnetic spectroscopy (MRS). MRS was performed using a General Electric 1.5T Signa EchoSpeed scanner (TR 3000 ms, TE 30 ms, PRESS). Absolute metabolite concentrations were determined using the LCModel fitting software. No significant reduction of NAA concentration was evident in the NAWM of the patients (patients: median 7.3 mM; controls: median 7.7 mM; P=0.19). There was, however, a significantly lower concentration of NAA in lesions (median 6.6 mM, P=0.015). Absolute values of choline-containing compounds, creatine and myo-inositol were significantly raised in the lesions (P=0.007, P=0.011 and P=0.002 respectively). The low NAA in lesions is consistent with axonal loss, damage or dysfunction occurring focally at the earliest clinical phase of the disease. The lack of any significant reduction in NAA in patient NAWM demonstrates that more widespread axonal changes are not yet detectable at this early clinical stage. A larger cohort and follow-up will be necessary to determine whether or not MRS findings have any prognostic significance for individual patients or sub-groups. This will also enable the clarification of the time course, pathogenesis and pathophysiological significance of the development of the low NAA, which is found in the NAWM of many patients with established MS.

Relative bioavailability of pilocarpine from a novel ophthalmic delivery system and conventional eyedrop formulations.

Pupillary responses to pilocarpine following topical application in a novel ophthalmic delivery system (NODS) and a conventional eyedrop formulation have been compared in eight healthy subjects in a single dose crossover study. The magnitude of the miotic and light reflex responses to NODS 40, 80, and 170 micrograms and to a single 2% Minims eyedrop (delivering 518 micrograms) were recorded by infrared television pupillography over periods of 24 hours. Dosage comparisons of drug responses were obtained by interpolation and yielded the equivalence of one eyedrop to 67 +/- 11 micrograms pilocarpine from the NODS formulation. These findings indicate that in a comparison of total doses delivered pilocarpine has an approximately eight-fold greater bioavailability from NODS than from a conventional eyedrop formulation.

Spectroscopic measurements in scleritis: bluish-red or deep red?

PURPOSE: To design a slit-lamp mountable spectrometer for the assessment of ophthalmic patients and to illustrate a potential clinical application by measuring the spectral characteristics of inflamed eyes of differing aetiologies. METHODS: A slit lamp mountable instrument was designed and built, and methods for data analysis developed. Reflectance spectra were recorded from two patients with scleritis, three with non-scleritic red eyes and from two controls with non-inflamed eyes. RESULTS: Measurements were reproducible and demonstrated statistically significant differences in the spectral characteristics between the three groups. Spectra from scleritic eyes revealed a relative increase in intensity of long wavelength red light, 650-740 nm, compared with non-scleritic red eyes. These longer wavelengths will be appreciated as dark red. There was no increase in relative intensity in the blue part of the spectrum in scleritic eyes. CONCLUSIONS: Reproducible measurements of the eye were made using an innovative, slit-lamp mountable spectrometer and its functionality demonstrated by differentiating the spectra from eyes with differing pathologies. While intending only to illustrate one potential application; for the cases examined, our results indicate that inflamed scleritic eyes exhibit a longer wavelength red light with no increase in shorter wavelength blue light. Thus our measurements would seem to confirm that the perceived redness of scleritis differs from other red eyes. However, it is a deeper darker red and not a bluish one as traditionally described.

Voluntary exercise can strengthen the circadian system in aged mice.

Consistent daily rhythms are important to healthy aging according to studies linking disrupted circadian rhythms with negative health impacts. We studied the effects of age and exercise on baseline circadian rhythms and on the circadian system's ability to respond to the perturbation induced by an 8 h advance of the light:dark (LD) cycle as a test of the system's robustness. Mice (male, mPer2(luc)/C57BL/6) were studied at one of two ages: 3.5 months (n = 39) and >18 months (n = 72). We examined activity records of these mice under entrained and shifted conditions as well as mPER2::LUC measures ex vivo to assess circadian function in the suprachiasmatic nuclei (SCN) and important target organs. Age was associated with reduced running wheel use, fragmentation of activity, and slowed resetting in both behavioral and molecular measures. Furthermore, we observed that for aged mice, the presence of a running wheel altered the amplitude of the spontaneous firing rate rhythm in the SCN in vitro. Following a shift of the LD cycle, both young and aged mice showed a change in rhythmicity properties of the mPER2::LUC oscillation of the SCN in vitro, and aged mice exhibited longer lasting internal desynchrony. Access to a running wheel alleviated some age-related changes in the circadian system. In an additional experiment, we replicated the effect of the running wheel, comparing behavioral and in vitro results from aged mice housed with or without a running wheel (>21 months, n = 8 per group, all examined 4 days after the shift). The impact of voluntary exercise on circadian rhythm properties in an aged animal is a novel finding and has implications for the health of older people living with environmentally induced circadian disruption.

Restricted wheel access following a light cycle inversion slows re-entrainment without internal desynchrony as measured in Per2Luc mice.

Circadian rhythms are physiological and behavioral oscillations that have period lengths of approximately 24 h. In mammals, circadian rhythms are driven by a master pacemaker in the hypothalamic suprachiasmatic nucleus (SCN). These rhythms can be entrained to light:dark cycles through photic and non-photic cues. Current research suggests that the SCN re-entrains rapidly to new light:dark (LD) cycles with the first photic cues, whereas peripheral tissues re-entrain more slowly, leading to a transient state of internal disorder while the organism adjusts to the new timing of photic input. To assess internal temporal order during the readjustment we used dim light to slow the rate of re-entrainment following a 12-h inversion of the LD cycle. We also used a wheel-restriction paradigm, which can block behavioral evidence of re-entrainment. Per2(Luc) mice were entrained to a 12:12 dim LD cycle with wheel access ad libitum. Following a 12-h shift in the LD cycle, some animals were subjected to wheel restriction; wheels were locked during the new dark period and available during the new light period. Other mice had wheels available ad lib throughout the experiment. Behavioral actograms of general locomotor activity as measured with motion sensors indicated that mice with ad lib access to wheels were able to re-entrain at a rate significantly faster than mice with restricted wheel access. Up to 2 weeks following the LD inversion many wheel-restricted animals were still active predominantly in the new light period. Phase of the PER2::LUC bioluminescence rhythms in SCN and four peripheral tissues (lung, esophagus, thymus, and spleen), measured ex vivo on days 2, 9, and 16 following the inversion, indicated that within each condition the SCN and peripheral tissues shifted at the same rate, whereas the rate of re-entrainment for the tissues differed between conditions. Ex vivo data showed that the PER2::LUC peaks in SCN and peripheral tissues were closely linked to time of activity onset in both groups. Thus, this wheel restriction protocol is capable of reducing and in some cases apparently hindering photic re-entrainment of the circadian system, verifying this protocol as a mechanism for study of photic/non-photic entrainment interactions. Our results suggest that LD inversion under dim light and a wheel-restriction protocol does not induce internal desynchrony, indicating that slowing the rate of shift by limiting both entrainment inputs may induce less "jet lag".

Sample sizes for brain atrophy outcomes in trials for secondary progressive multiple sclerosis.

BACKGROUND: Progressive brain atrophy in multiple sclerosis (MS) may reflect neuroaxonal and myelin loss and MRI measures of brain tissue loss are used as outcome measures in MS treatment trials. This study investigated sample sizes required to demonstrate reduction of brain atrophy using three outcome measures in a parallel group, placebo-controlled trial for secondary progressive MS (SPMS). METHODS: Data were taken from a cohort of 43 patients with SPMS who had been followed up with 6-monthly T1-weighted MRI for up to 3 years within the placebo arm of a therapeutic trial. Central cerebral volumes (CCVs) were measured using a semiautomated segmentation approach, and brain volume normalized for skull size (NBV) was measured using automated segmentation (SIENAX). Change in CCV and NBV was measured by subtraction of baseline from serial CCV and SIENAX images; in addition, percentage brain volume change relative to baseline was measured directly using a registration-based method (SIENA). Sample sizes for given treatment effects and power were calculated for standard analyses using parameters estimated from the sample. RESULTS: For a 2-year trial duration, minimum sample sizes per arm required to detect a 50% treatment effect at 80% power were 32 for SIENA, 69 for CCV, and 273 for SIENAX. Two-year minimum sample sizes were smaller than 1-year by 71% for SIENAX, 55% for CCV, and 44% for SIENA. CONCLUSION: SIENA and central cerebral volume are feasible outcome measures for inclusion in placebo-controlled trials in secondary progressive multiple sclerosis.

Evaluating clinical outcome and staff morale in a rehabilitation team for people with serious mental health problems.

Tameside and Glossop rehabilitation team (in England) have developed a progressive and targeted service for people with serious mental health problems through the systematic implementation of research-based evidence in practice and service configuration. This study was undertaken to provide a method of auditing the clinical outcome of the service and monitoring staff morale in a manner which could be integrated in the day to day delivery of services, and which could inform future service developments. Changes in the functioning of the total population of rehabilitation team clients were assessed over a 1-year period by Health of the Nation Outcome Scales (HoNOS) ratings at 6-monthly intervals. Factors causing stress and stress levels among all staff were assessed using the Mental Health Stress Questionnaire. The findings give clear indications of areas of the service which needed improving or changing, and identify ways in which the ongoing process of data collection might be refined.