Abstract: Acute myeloid leukemia (t-AML) is one of the worst adverse events of mitoxantrone treatment, but the exact risk in multiple sclerosis (MS) patients is not yet known. We describe a case wherein the patient developed t-AML 11 months after mitoxantrone had been discontinued. The patient was treated by polychemotherapy and autologous bone marrow transplantation with complete recovery of t-AML and stabilization of the neurological disease.

Abstract: Therapeutic proteins like recombinant human interferon-beta (rhIFNbeta) may induce neutralising antibodies (NABs), which inhibit their efficacy. Hence, there is a great need for strategies to predict whether a formulation will induce an immune response. Immune tolerant transgenic animals are important tools to study this phenomenon. This article describes a bioassay for NABs detection in mouse sera. The bioassay corresponds to the MxA Gene expression Assay (MGA) for human sera and measures the inhibition by mouse serum of the IFNbeta induced MxA mRNA. Samples from 6 non-immunised and 14 IFNbeta-immunised mice were tested for both binding antibodies (BABs) and NABs using the bioassay. All 16 mouse sera tested positive for NABs were also positive for BABs; BAB and NAB levels were correlated with a coefficient of 0.62 (p=0.0186). The intra-assay variations ranged from 1.38% to 5.26% (mean 3.03%). Effects of cytotoxicity against A549 cells were slightly evident at low serum dilutions (i.e. 1/10, 1/20), but levels of damaged cells were easily evaluated based on the threshold cycle (Ct) values of the housekeeping gene 18SrRNA. The possibility of measuring NABs, in addition to BABs, in mouse sera increases the usefulness of the animal model, in studying the many factors influencing the immunogenicity of rhIFNbeta.

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Abstract: BACKGROUND: The cytokine interferon beta (IFNbeta) is successfully used in the treatment of multiple sclerosis (MS), although there is a high degree of variability in the response. A common mechanism involved in the modulation of responsiveness to cytokines is represented by regulation of their receptor expression through autocrine ligand-mediated loops. The present study is aimed at investigating the regulation of IFNalpha/beta receptor (IFNAR) during IFNbeta therapy in patients with MS and at correlating it with the biologic responsiveness to the cytokine. METHODS: Quantitative PCR measurements of IFNAR-1 and the three IFNAR-2 isoforms were performed in 141 patients after short-term and long-term treatment. Patients were also regularly screened for anti-IFNbeta neutralizing antibodies (NAbs). IFN-inducible myxovirus resistance protein A messenger RNA was used as an indicator of bioactivity. RESULTS: Pretreatment levels of IFNAR-2 in patients were lower overall than in controls (p = 0.038), and high levels correlated with greater bioactivity. Upon prolonged treatment, NAb-negative patients displayed a state of decreased transmembrane IFNAR-2 expression (p </= 0.025), whereas levels of soluble IFNAR-2 were slightly increased (p < 0.0001). The presence of NAbs reversed these effects (p </= 0.0056). In NAb-positive patients, pretreatment expression levels of both transmembrane IFNAR-2 isoforms were significantly lower than in NAb-negative patients (p </= 0.0089). CONCLUSIONS: Findings show that interferon-alpha/beta receptor (IFNAR)-2 isoforms are important regulators of the responsiveness to endogenous and systemically administered interferon beta (IFNbeta). They show a dual action, agonistic and antagonistic, that influences both the magnitude and the nature of the biologic response to IFNbeta. Levels of IFNAR-2 are regulated with the aim of keeping the body in a state of equilibrium, even when nonphysiologic stimuli are present.

Abstract: BACKGROUND: Evidence is accumulating that indicates that a selected assessment of gray matter (GM) damage is able to provide strong paraclinical correlates of multiple sclerosis (MS) severity. OBJECTIVE: To investigate the pattern of regional GM atrophy in patients with benign MS (BMS) vs those with secondary progressive MS (SPMS) to better elucidate the factors associated with a favorable status in patients with MS. DESIGN: Cross-sectional survey from January 2006 to August 2007. SETTING: Referral, hospital-based MS clinics. Patients Sixty patients with BMS, 35 patients with SPMS, and 21 healthy volunteers. MAIN OUTCOME MEASURES: Neuropsychological tests exploring memory, attention, and frontal lobe cognitive domains were administered to BMS patients. A voxel-based morphometry analysis of GM concentration was performed using statistical parametric mapping and a threshold of 0.05, corrected for multiple comparisons. RESULTS: Twelve BMS patients (20%) had an abnormal performance on 3 or more neuropsychological tests. Compared with healthy individuals, BMS patients had a reduced GM volume in the subcortical and frontoparietal regions. Compared with BMS patients, those with SPMS had a significant GM loss in the cerebellum. No differences between BMS and SPMS patients were found when only BMS patients with cognitive impairment or those with shorter disease duration (15-19 years) and higher Expanded Disability Status Scale scores (>2.0) were considered. CONCLUSIONS: Cerebellar GM atrophy seems to be a major determinant of irreversible locomotor disability in MS. The absence of cognitive impairment and a longer disease duration or lower Expanded Disability Status Scale score may identify those BMS patients with the potential for a favorable disease evolution.

Abstract: BACKGROUND: Prolonged therapy with interferon beta (IFN beta) often leads to the development of anti-IFN beta binding antibodies (BAbs). A subset of the BAbs is of a neutralizing nature (neutralizing antibodies, NAbs) and is associated with reduced clinical efficacy of therapy. Myxovirus-resistance-protein A (MxA) has proven to be a reliable biomarker of IFN beta bioactivity. We analyzed the prognostic value of MxA mRNA, NAbs, and BAbs on the risk of having a new relapse in IFN beta-treated patients. METHODS: A 3-year study was conducted in 137 IFN beta-treated patients. Blood samples for BAbs, NAbs, and MxA mRNA measurements were taken after 12 +/- 3 months of therapy. Analysis of relapse-free survival (RFS) was performed for all measures by using known thresholds, generating "positive" and "negative" groups. Also, time between sampling and following relapse and risk of new relapses were calculated. RESULTS: The MxA-negative group showed poorer RFS rates than the MxA-positive group [p < 0.0001, hazard ratio (HR) = 2.87]. Likewise, the NAb-positive group showed poorer RFS rates than the NAb-negative group (p =0.0013; HR = 2.49). On the contrary, BAb measurement did not show a clear clinical significance. CONCLUSIONS: Findings indicate that measurements of both myxovirus-resistance-protein A (MxA) and neutralizing antibodies (NAbs) predict the risk of new relapses; however, the slightly stronger prognostic significance of MxA mRNA and the easier method for it measurement make MxA mRNA the preferred biomarker for monitoring interferon beta (IFN beta)-treated patients. This information can be used to better tailor treatment to the individual patient with MS.

Abstract: BackgroundNeutralising antibodies (NAb) to interferon beta (IFNbeta) are associated with a reduced bioactivity and efficacy of IFNbeta in multiple sclerosis (MS). Unclear is how to apply IFNbeta bioactivity measurements (quantification of Myxovirus resistance protein A (MxA) mRNA) in clinical practice.ObjectivesTo evaluate value and feasibility of IFNbeta bioactivity measurement with a single MxA mRNA measurement for screening and a second measurement before and after IFNbeta administration for definite confirmation of IFNbeta bioactivity status.MethodsIn 79 MS patients MxA mRNA expression was determined 4 hours after IFNbeta administration. If inadequate, MxA mRNA expression testing was repeated 3 months afterwards, comparing post- and pre injection samples to determine whether IFNb bioactivity was persistently lacking. MxA mRNA expression was compared to NAbeta titres, determined by the cytopathic effect assay (CPE).ResultsNAb titres correlated significantly with MxA mRNA expression and MxA mRNA induction. Of all screened patients, only one patient had adequate MxA mRNA expression and high NAb titres simultaneously. Of the biological non-responders at second measurement (21/55), 17 (81%) were high-titre NAb positive, 1 (5%) was low-titre NAb positive and 3 (14%) were NAb negative. Without considering the pre-injection measurement, two more NAb negative patients would have tested negative for IFNbeta bioactivity, emphasizing the need of a pre-injection sample.ConclusionsOur data suggest that for IFNbeta bioactivity screening a single post-injection measurement seems reasonable. However, MxA induction measurement based on both pre- and post-IFNbeta injection samples at second measurement is somewhat more precise in determining ultimate IFNbeta bioactivity status.

Abstract: Neutralising antibodies develop in 15% of interferon-beta (IFNbeta)-treated patients, causing the reduction of the clinical effects of the treatment. This is the first study that shows that switching patients from IFNbeta to glatiramer acetate (GA) in case of neutralising antibodies (NAb) positivity is effective in reducing relapse rate and in delaying the time to first relapse. In conclusion, our data suggest the use of GA in NAb-positive patients.

Abstract: The therapeutic benefits of interferon-beta are limited, as some patients with multiple sclerosis (MS) do not respond to therapy. Based on their biological characteristics, non-responsive patients can be divided into three subgroups: genetic, pharmacological and pathogenetic non-responders. In order to tailor the best treatment in both newly diagnosed MS patients and those already receiving treatment, the neurologist must carefully consider the risk of treating non-responders.

Abstract: OBJECTIVE: Although in benign multiple sclerosis (BMS) locomotor disability is absent or only minimal, subclinical cognitive impairment seems to occur in many cases. Diffusion tensor (DT) MRI enables us to quantify the extent of "actual" tissue damage, which goes undetected when using conventional MRI. Against this background, we investigated the extent of structural brain damage underlying cognitive dysfunction in BMS, with the ultimate aim to move a first step toward a more reliable definition of this disease phenotype. METHODS: Conventional and DT MRI scans of the brain were acquired from 62 BMS patients. Thirty-six secondary progressive multiple sclerosis (SPMS) patients and 19 healthy subjects served as controls. In BMS patients, neuropsychological tests exploring memory, attention, and frontal lobe functions were administered. Normalized brain volume (NBV), mean diffusivity (MD), and fractional anisotropy (FA) of the normal-appearing white matter (NAWM) and MD of the gray matter (GM) were computed. RESULTS: Twelve BMS patients (19%) fulfilled predefined criteria for cognitive impairment. BMS patients had abnormal MD and FA values from both NAWM and GM. Whereas BMS patients without cognitive impairment had lower T2 LV (p = 0.03), higher NBV (p = 0.006), and lower average GM MD (p = 0.03) than SPMS patients, BMS patients with cognitive impairment did not significantly differ from SPMS patients for any MRI-derived metric. CONCLUSIONS: In benign multiple sclerosis (BMS), cognitive dysfunction is associated with severe structural brain damage, which resembles that of patients with a much more disabling disease course. A reliable definition of BMS should, therefore, include the preservation of cognitive functioning as an additional requisite.

Abstract: Interferon beta (IFNbeta) therapy for multiple sclerosis (MS) is associated with a potential for the development of neutralising antibodies (NAbs) that negatively affect therapy. Several factors influence the development of NAbs, such as lack of complete sequence homology with the endogenous IFNbeta sequence, frequency of administration, level of dose and formulation of IFNbeta. Taken together, the evidence that NAb status reduces clinical efficacy in MS patients is strong. Standardised assays for NAbs are lacking, and titres vary over time. NAb testing is a critical component of care for MS patients because it provides information on one of the most important factors determining clinical responsiveness to IFNbeta therapy. This expert panel report attempts to move the field towards resolution of the remaining issues and considers several aspects of NAbs, including their clinical relevance, factors influencing immunogenicity, assays to quantify NAbs and the definition of clinically relevant titres.

Abstract: There are two commonly employed types of bioassays for the detection of neutralizing antibodies (NAbs) against interferon-beta (IFNbeta): the cytopatic effect assay (CPE), and the MxA (myxovirus resistance protein A) protein assay (MPA). This article describes a bioassay based on the real time PCR measurement of mRNA that results from the induction, in cultured human cells, of the MxA gene by IFNbeta. Serum samples from 104 patients with multiple sclerosis (MS) treated with IFNbeta were tested for NAbs using our real time PCR bioassay. NAbs also were measured in the same specimens by the MPA assay and CPE assay. The calibration range of the real time PCR bioassay is 0.125-30 LU/mL. The range of the intra- and inter-assay variations (coefficients of variation in log(10)) were 4.05% (range 0.88%-7.90%) and 4.42% (range 0.31%-9.15%), respectively. Samples of the three commercial preparations of IFNbeta-1a and -1b were measured showing dose-response curves parallel to that of the NIH reference IFNbeta (mean SD at the midpoint of the dose-response curve=5%). In addition, the assay was robust with respect to number of cells plated (i.e., increasing cell densities from 12x10(3)/well to 384x10(3)/well resulted in 3.03% variability in MxA expression normalized with glyceraldehyde-3 phosphate dehydrogenase). NAbs titers measured were closely comparable to those obtained by the MPA [r(spearman)=0.899; 89% of observed agreements; K=0.779] and the CPE [r(spearman)=0.7899); 86%; K=0.729] assays. Despite the obvious disadvantage of cost, when carried out according to quality assurance guidelines for molecular diagnostics the new MxA gene-expression assay (MGA) has significant advantages over the other methods for testing NAbs: it has excellent reliability and reproducibility, and utilizes equipment and methodologies already accessible in many clinical laboratories.

Abstract: BACKGROUND: Brain atrophy, as assessed by magnetic resonance imaging (MRI), has been correlated with disability in patients with multiple sclerosis (MS). Recent evidence indicates that both white matter (WM) and gray matter (GM) are subject to atrophy in patients with MS. Although neurological deficiencies in MS are primarily due to loss of WM, the clinical significance of GM atrophy has not been fully explored in MS. METHODS: We have undertaken a three-year, open-label study, comparing 26 patients who elected to receive intramuscular interferon beta-1a (IFN beta-1a) therapy, with 28 patients who elected not to receive therapy. Both groups had quantitative cranial MRI scans at study entry and after three years, and standardized clinical assessments every six months. Brain parenchymal fraction (BPF), GM fraction (GMF), and WM fraction (WMF) percent changes were calculated, and T2- and T1-lesion volumes (LVs) assessed. RESULTS: After three years, mean percent (%) change in BPF favored the IFN beta-1a treatment group (IFN beta-1a -1.3% versus the control group -2.5%, P=0.009), as did the mean percent change in GMF (+0.2 versus -1.4%, P=0.014), and the mean percent change in T1-LV (-9.3 versus +91.6%, P=0.011). At the end of the study, there was a significant within-patient decrease in BPF for both groups (P=0.02 for the IFN beta-1a treatment group, and P<0.001 for the control group), a significant within-patient decrease in WMF for the IFN beta-1a treatment group (P=0.01), and a significant decrease in GMF for the control group (P=0.013) when compared with baseline. CONCLUSION: Over a three-year period, treatment with IFN beta-1a significantly slowed the progression of whole-brain and GM atrophy, and of T1-hypointense LV accumulation, when compared with the control group.

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Abstract: Many multiple sclerosis (MS) patients treated with interferon-beta (IFNbeta) develop anti-IFNbeta antibodies (BAbs), which can interfere with both in vitro and in vivo bioactivity of the injected cytokine. Objective of this study was to correlate these measures. Among the 256 enrolled patients, 11 (4.3%) showed a significant inhibition of the IFNbeta activity in vitro, but no measurable BAbs. As a whole, in vivo bioactivity was inhibited in 9/11 (82%) of these patients. A minority of IFNbeta treated patients have a non-antibody mediated neutralising activity, which competitively inhibits the bioactivity both in vitro and in vivo.

Abstract: We have described two cases of Devic's disease patients treated with rituximab with different outcomes. The results indicate that there may be early unresponsiveness in very aggressive cases. Well designed clinical trials are needed to assess treatment effects in such a rare disease.

Abstract: Cervical cord damage is likely to contribute to the accumulation of disability in multiple sclerosis (MS) and can be quantified in vivo using MRI. We used conventional and diffusion tensor (DT) MRI to: (a) define the temporal evolution of intrinsic tissue injury and atrophy in the cervical cord from MS patients, (b) investigate how these two aspects of cord damage are interrelated and (c) assess the correlation of cord MRI metrics with concomitant brain damage and disability. Conventional and DT MRI of the brain and cervical cord were obtained from 42 MS patients and 9 healthy controls at baseline and after a mean follow-up of 2.4 years. At each time-point, we measured: cervical cord lesion number, cross-sectional area, mean diffusivity (MD) and fractional anisotropy (FA). Brain T2 lesion volume, grey matter MD, normal appearing white matter (NAWM) MD and FA, as well as longitudinal normalized percentage brain volume changes were also measured. In MS patients, cervical cord cross-sectional area (P < 0.001) and FA (P = 0.01) decreased, and cervical cord MD increased (P < 0.001) during follow-up. Cord FA decrease, but not cord cross-sectional area and MD, was significantly higher (P = 0.05) in primary progressive MS patients than in those with either relapsing-remitting or secondary progressive MS. At baseline and follow-up, moderate correlations were found between intrinsic cord diffusivity abnormalities and cord cross-sectional area (r values ranging from 0.34 to 0.58), but not between their changes over time. No cross-sectional and longitudinal correlations were found between these MRI metrics and the number of cord T2-visible lesions. Brain NAWM MD (P = 0.03) and brain volume (P < 0.001) also changed in patients. There was no significant correlation between cord and brain MRI metrics at both time-points, as well as between their changes occurred over the follow-up. Baseline cord cross-sectional area (r = -0.40, P = 0.01) and FA (r = -0.40, P = 0.03) correlated with increase in disability at follow-up. This study shows that both progressive tissue loss and injury to the remaining tissue occur in the cervical cord of MS patients, and that these two components of cord damage are not strictly interrelated, thus suggesting that a multiparametric MRI approach is needed to achieve more accurate estimates of such a damage. MS cord pathology also seems to be independent of concomitant brain changes, to develop at different rates according to disease phenotype, and to be associated to medium-term disability accrual.

Abstract: OBJECTIVE: Glucocorticoids (GCs) at pharmacological doses stimulate bone resorption. Mechanisms of this action are unclear. The osteoclastogenic cytokine interleukin (IL)-6 acts through an oligomeric receptor consisting of two subunits, gp80 (or IL-6 receptor alpha, IL-6Ralpha) and gp130; both exist in membrane and soluble forms. Soluble IL-6Ralpha (sIL-6Ralpha) enhances, while sgp130 inhibits IL-6 signalling. In vitro, GCs enhance many effects of IL-6 by up-regulation of IL-6Ralpha. The aim of the present study was to assess acute changes of IL-6 system in the peripheral blood of patients given high-dose GCs. SUBJECTS AND METHODS: Serum levels of IL-6, sIL-6Ralpha, sgp130 and bone turnover markers were assessed before and each day during treatment in 24 multiple sclerosis (MS) patients undergoing high-dose (prednisolone, 15 mg/kg per day), short-term (3 to 5 days) intravenous GC therapy for relapse at the Regional Multiple Sclerosis Centre. RESULTS: An immediate and marked fall of osteocalcin and an early increase of C-terminal telopeptide of type I collagen were already noticed at day 2 (P < 0.001 and P < 0.02, respectively); both became more apparent in the subsequent days. IL-6 was always below or near the detection limit of our ELISA. sgp130 showed a slight increase. sIL-6Ralpha significantly increased, peaking at day 4 (P < 0.01). However, inter-individual variability of response was noticed. Four patients showed a slight decrease, while no change was observed in one patient and an increase was noticed in the remaining nineteen (maximum change ranging from +10% to +67% with respect to baseline). In these patients, a significant increase of sIL-6Ralpha/sgp130 ratio was apparent. No correlation was found between bone turnover markers and any measured component of the IL-6 system. CONCLUSIONS: sIL-6Ralpha and sIL-6Ralpha/sgp130 ratio are precociously increased in the peripheral blood of the vast majority of patients given high-dose, intravenous GCs. The increase of systemically available sIL-6Ralpha conceivably results in the enhancement of IL-6-dependent osteoclastogenesis. The role of such a mechanism in the bone loss observed in inflammatory and immune-mediated diseases (where abundancy of IL-6 in the bone microenvironment is expected) requires further investigation.

Abstract: Biological activity of interferon-beta (IFNbeta) can be assessed by measuring IFN-stimulated genes (ISGs). Among them, myxovirus resistance protein A (MxA) appears to have the highest specificity, but it has no role in the pathogenesis of multiple sclerosis (MS). To investigate the reliability of MxA as a biomarker, we compared its expression to that of two other ISGs: TNF-related apoptosis-inducing ligand (TRAIL) and X-linked inhibitor of apoptosis factor-1 (XAF-1). Both were shown to be involved in immunoregulatory mechanisms and might play a role in MS. Quantitative-PCR measurements were performed in peripheral blood mononuclear cells from 73 MS patients after short-term and long-term treatment with IFNbeta. A time-dependent response for multiple ISGs was observed in all patients after short-term treatment. In contrast, long-term treatment induced concurrent inhibition of ISGs in 12.3% (9/73) of patients, in whom neutralizing antibodies (NAbs) were detectable. Besides, 22% (16/73) of chronically treated patients showed a non-NAbs-related abrogation of TRAIL expression. In summary, 1) MxA expression was significantly higher than both TRAIL and XAF-1, and 2) MxA was the most sensitive gene to detect decreased bioavailability due to NAbs. These findings identify MxA as an appropriate biomarker for IFNbeta, although there is no evidence for a functional role of it in MS.

Abstract: Reliable prognostic markers of primary progressive (PP) multiple sclerosis evolution are still needed. Diffusion tensor (DT) MRI can quantify normal-appearing white matter (NAWM) and grey matter (GM) damage in multiple sclerosis patients. We investigated whether conventional and DT-MRI-derived measures can predict the long-term clinical evolution of PP multiple sclerosis. In 54 PP multiple sclerosis patients, conventional and DT-MRI scans of the brain and T1-weighted scans of the cervical cord were acquired at baseline and after a median follow-up of 15 months. Another clinical evaluation was performed, 56 months after baseline, in 52 patients. Measures of lesion load, brain and cord atrophy were obtained. Histograms of the mean diffusivity (MD) and fractional anisotropy (FA) values from the NAWM and GM were analysed. At follow-up, 35 patients (65%) experienced a confirmed disability progression. Baseline expanded disability status scale score and average GM MD were independent predictors of subsequent clinical deterioration in a multivariable model (Nagelkerke R2: 0.44; discriminating ability: 81%). A lower level of disability and a more severe GM damage identify PP multiple sclerosis patients with an increased risk of disease progression over the subsequent 5 years. These data may be relevant to select patients for future exploratory phase II trials.

Abstract: To date, inter- and intra-laboratory consistency of binding assays for measuring anti-interferon (IFN)beta antibodies has not been assessed. In this investigation, two independent laboratories tested a library of 80 serum specimens obtained from multiple sclerosis (MS) patients treated with IFNbeta. For binding antibodies (BAbs) evaluations, each laboratory used both a capture-ELISA (cELISA) and an enzyme-immuno-assay (EIA), which is commercially available. Samples were also tested for neutralizing antibodies (NAbs). Data demonstrated good intra-laboratory reliability (r(pearson) > or = 0.86), and a good overall agreement between the results obtained from the two centers, using both the cELISA (69/80 of observed agreements) and the EIA (67/80). Accordingly, kappa coefficients (K) showed good concurrence (K > or = 0.651). There was also substantial agreement between cELISA and EIA measurements, as performed in both centers (Orbassano, 66/80, K = 0.631; Basel, 70/80, K = 0.717). However, by comparing NAbs and BAbs titers obtained with both assays, we found that a high degree of BAb-negative samples were positive in NAb-assay. Thus, our study does not support the usefulness of ELISA-based BAb assays as a screening tool for NAbs. Otherwise, BAb-assays can be used as a confirmation test, indicating that the decrease of the biological effects is due to antibodies. In this context, both ELISA-based assays are equally reliable techniques.

Abstract: The mechanisms underlying the progressive course of multiple sclerosis (MS) are not fully understood yet. Since diffusion tensor (DT) MRI can provide quantitative estimates of both MRI-visible and MRI-occult brain damage related to MS, the present study investigated the value of DT MRI-derived measures for the assessment of the short-term accumulation of white and gray matter (GM) pathology in patients with primary progressive (PP) and secondary progressive (SP) MS. Fifty-four patients with PPMS and 22 with SPMS were studied at baseline and after a mean follow-up of 15 months. Dual-echo, T1-weighted, and DT MRI scans of the brain were acquired on both occasions. Total lesion volumes (TLV) and percentage brain volume changes (PBVC) were computed. Mean diffusivity (MD) and fractional anisotropy (FA) maps of the normal-appearing white (NAWM) and gray matter (NAGM) were produced, and histogram analysis was performed. In both patient groups, a significant increase of average lesion MD (P = 0.01) and of average NAGM MD (P = 0.007) was found at follow-up. No significant differences between PPMS and SPMS patient groups were found for the on-study changes of any MRI-derived measure. No significant correlations were found between the percentage changes of DT MRI-derived measures and those of TLV and PBVC. No significant changes of DT MRI-derived measures were observed in age-matched healthy controls over the same study period. Over a 1-year period of follow-up, DT MRI can detect tissue changes beyond the resolution of conventional MRI in the NAGM of patients with progressive MS. The accumulation of DT MRI-detectable gray matter damage does not seem to merely depend upon the concomitant increase of T2-visible lesion load and the reduction of brain volume. These observations suggest that progressive NAGM damage might yet be an additional factor leading to the accumulation of disability in progressive MS.

Abstract: This study is the first to evaluate biological response to first injections of interferon-beta (IFNbeta) in patients with multiple sclerosis. MxA mRNA was measured in 96 patients receiving IFNbeta-1a (Avonex, n=32), IFNbeta-1b (Betaferon, n=19), IFNbeta-1a (Rebif) 22 microg (n=30), or IFNbeta-1a 44 microg (n=15). Patients were analysed before, 3 and 24 h after the first injection, and 12 h after the second administration. Results showed that up-regulation was evident within 3 h of IFNbeta injection, peaked 12 h after injection, and progressively declined 24 h after administration. The cumulative responses were similar after a single administration, regardless of product/dose. Moreover, data indicate that the abolition of the biological activity detected during IFNbeta therapy is due to underlying phenomena (e.g., neutralizing antibodies), because all patients were constitutively responders to IFNbeta at treatment initiation.

Abstract: Aggressive forms of multiple sclerosis (MS) represent a limited group of demyelinating diseases that rapidly progress to severe disability. Currently available therapies are poorly effective against these clinical entities. Recently, it has been demonstrated that intense immunosuppression followed by autologous haematopoietic stem cell transplantation (AHSCT) can affect the clinical course of individuals with severe MS and completely abrogate the inflammatory activity detected by MRI. We report the result of the Italian phase 2 GITMO study, a multicentre study in which 21 MS patients, who were rapidly deteriorating and not responding to the usual therapeutic strategies, were treated with this procedure. The clinical effect of the treatment is long lasting, with a striking abrogation of inflammation detected by MRI findings. These results support a role for intense immunosuppression followed by ASCT as treatment in rapidly evolving MS cases unresponsive to conventional therapies.

Abstract: Relapsing-remitting multiple sclerosis (MS) has a very fluctuating course and responsiveness to interferon beta (IFN-beta) treatment in each patient is extremely difficult. Agreement exists about the negative role of neutralising antibodies (NAbs) on clinical efficacy and markers of IFN-beta bioavailability have been studied; no guidelines exist yet about what to do when a patient becomes NAbs positive or IFN biological activity is lost. In this study 405 MS patients have been longitudinally studied for NAbs and MxA expression. A spontaneous disappearance of NAbs was observed in a few patients with low antibody titre; according to the clinical course, a therapeutic modification has been made in patients persistently NAbs positive; in these patients NAbs persisted over time despite the interruption of IFN therapy.

Abstract: PURPOSE OF REVIEW: Antibodies against interferon-beta (IFN-beta) can appear in a relevant number of patients. The subset of antibodies that can neutralize IFN-beta activity are called neutralizing antibodies. This review focuses on their impact both on therapeutic efficacy and on bioactivity of IFN-beta, and on the management of antibody-positive patients. RECENT FINDINGS: When IFN-betas were first used, neutralizing antibodies were not considered important. However, recent clinical, biologic, and immunologic data have demonstrated that they reduce or abolish the therapeutic efficacy of IFN-beta in 10-20% of patients. Quantification of antibodies using various biologic methods make it difficult to compare among different laboratories, and hence, standardization of assay procedures is necessary. Despite these technical difficulties, data consistently show differences in immunogenicity among the different IFN-beta products and the negative effects of neutralizing antibodies on the clinical efficacy of IFN-betas. Because the therapeutic action of IFN-beta depends on activation of IFN-inducible genes, new methods for the quantification of the biologic activity of IFN-beta have been developed, and a good correlation has been found between the presence of neutralizing antibodies and abrogation of IFN-beta bioactivity. SUMMARY: Quantification of neutralizing antibodies and the in-vivo bioactivity of IFN-beta through IFN-beta-inducible gene products such as Myxovirus protein A, offer valuable information on IFN-beta therapy. Important questions such as the optimal therapeutic strategy for managing neutralizing antibodies positive patients require further study in clinical trials.

Abstract: Neutralizing antibodies (NAb) against interferon-beta (IFN-beta) develop in about a third of treated multiple sclerosis patients and are believed to reduce therapeutic efficacy of IFN-beta on clinical and MRI measures. The expression of the interferon acute-response protein, myxovirus resistance protein A (MxA) is a sensitive measure of the biological activity of therapeutically applied IFN-beta and of its reduced bioavailability due to NAb. However, MxA may not be operative in the pathogenesis of multiple sclerosis or the therapeutic effect of IFN-beta. Instead, matrix metalloproteinases (MMPs) are increased in brain tissue, CSF and blood circulation of multiple sclerosis patients and function as effector molecules in several steps of multiple sclerosis pathogenesis. One of the molecular mechanisms by which IFN-beta exerts its beneficial effect in multiple sclerosis is reduction of MMP-9 expression and increase of its endogenous tissue inhibitor, TIMP-1. Quantitative PCR measurements of MMP-2 and MMP-9, TIMP-1 and TIMP-2, and MxA were performed in peripheral mononuclear cells from clinically stable multiple sclerosis patients with relapsing remitting disease course after short-term and long-term treatment with IFN-beta. IFN-beta therapy down-regulated the expression of MMP-9 and abolished that of MMP-2 in long-term, but not short-term treated multiple sclerosis, while levels of MxA were increased in both instances. The presence of NAb reversed these effects, i.e. led to reduced MxA and increased MMP-2/MMP-9 expression levels compared with NAb- patients. In contrast, expression of TIMPs in peripheral blood mononuclear cells remained unaffected by IFN-beta therapy and the presence of NAb. While MxA is able to detect the biological action and reduced bioavailability of IFN-beta on the basis of single injections, only MMP-9 shows quantitative correlation with the NAb titre. Together with evidence that an imbalance between MMP and TIMP expression is a crucial pathogenetic feature in multiple sclerosis, these findings support the concept of a significant role of NAb in reducing the therapeutic efficacy of IFN-beta.

Abstract: Protein-based therapies are useful in a variety of diseases; however, their potential for immunogenicity is a disadvantage. Neutralizing antibodies (NAbs) that develop to interferon beta (IFNbeta) products (IFNbeta-1b, IFNbeta-1a-Avonex((R)), or IFNbeta-1a-Rebif((R))), which are first-line therapies for the treatment of multiple sclerosis, are reported to reduce the clinical efficacy of these agents. In individual clinical studies of each commercially available IFNbeta product, 28% to 47% of patients develop NAbs to IFNbeta-1b, 12% to 28 % to IFNbeta-1a-Rebif, and 2% to 6% to IFNbeta-1a-Avonex. Problems exist in comparing the incidence of NAbs among IFNbeta products across studies because of differences in study methodology, including assay methods, treatment duration, and the definition of NAb positive. Results from studies that have directly compared these products are consistent with results from the respective clinical trials of IFNbetas. Both the clinical trials and the independent studies have shown that NAbs develop more frequently with IFNbeta-1b treatment than with IFNbeta-1a treatment and that, among IFNbeta-1a products, NAbs develop more frequently with IFNbeta-1a-Rebif treatment than with IFNbeta-1a-Avonex treatment. Factors that may affect the immunogenicity of IFNbetas, including the dosing regimens and the biochemical properties of the products, are discussed.

Abstract: BACKGROUND: Interferon beta (INFbeta) may induce the expression of several proteins, including neopterin, considered a biological marker of INFbeta activity. OBJECTIVES: The aim of this study was to determine the serum neopterin concentration at the beginning of, and during, IFNbeta-1a therapy in relapsing-remitting multiple sclerosis (r-r MS) patients, and to look for a possible correlation between protein synthesis and the clinical course of the disease. METHODS: Thirteen r-r MS patients were treated with INFbeta-1a (i.m. 6 MIU/week) for 2 years. Blood samples for neopterin determinations were taken daily over a period of 1 week at the end of each 6 months of therapy, and tested for neutralizing antibodies (NABs). RESULTS: Neopterin levels peaked 24-48 h post-injection and returned to baseline after 120 h. After 1 year of therapy, four patients dropped out of the study because of progression of the disease: in these subjects a significant decrement of neopterin was observed. CONCLUSION : Neopterin baseline values were not found to decrease over the 2 years of therapy, and neopterin may be considered to be a useful marker of responsiveness to IFNbeta.

Abstract: OBJECTIVE: To analyze the impact of neutralizing antibodies (NAbs) on the clinical efficacy of IFNbeta. METHODS: This was an open-label study involving 78 patients with multiple sclerosis treated with Betaferon 8 million IU (MIU) subcutaneously (SC) every other day (n = 20), Rebif 22 micro g SC 3 times weekly (n = 25), or Avonex 30 micro g IM once weekly (n = 33). Every 3 months, blood samples were collected and sera were analyzed for NAbs using an antiviral cytopathic effect assay. Patients were categorized according to their NAb status: NAb negative (NAb-); isolated NAb positive (NAb+), patients with > or =1 positive sample (titer > or = 20); and persistent NAb+, patients with > or =2 consecutive positive samples (titer > or = 20). Patients who were NAb- and persistent NAb+ were compared for relapse rate, time between first and second relapse, percentage of relapse-free patients, and percentage of patients who had a sustained progression of > or =1 point on the Expanded Disability Status Scale (EDSS). RESULTS: The incidence of persistent NAb+ patients was 35% for Betaferon, 20% for Rebif, and 3% for Avonex. During IFNbeta treatment, both NAb+ and NAb- patients showed a reduction in relapse rate; this reduction (25%) was not significant in NAb+ patients but was significant (67%; p < 0.0001) in NAb- patients. In addition, the mean relapse rate was higher (p = 0.039), mean time between first and second relapse was shorter (13 vs 21 months; p = 0.0064), and there was a trend suggesting that NAbs affected the probability of remaining relapse free (p = 0.08). A higher percentage of NAb+ patients versus NAb- patients had worsening of EDSS scores during follow-up (p = 0.013). CONCLUSION: Persistent NAbs significantly reduce the clinical efficacy of IFNbeta.

Abstract: Treatment of multiple sclerosis (MS) with interferon beta (IFNbeta) can be associated with the development of binding antibodies (BAbs) and neutralizing antibodies (NAbs). NAbs are a subset of BAbs that prevent IFNbeta from effectively binding to or activating its receptor, thereby blocking its biologic effects and inhibiting its therapeutic effects. Several factors can affect the incidence and titers of NAbs that develop to IFNbeta, including the type of IFNbeta preparation used for treatment. One of the major limitations to evaluating the relative importance of these factors is the variation in assays used to detect IFNbeta antibodies. Two major types of assays are used to detect antibodies to IFNbetas: [1] binding assays, which measure the ability of antibodies in patients' sera to bind to IFNbeta; and [2] neutralization assays (or bioassays), which measure the ability of patients' sera to neutralize the biologic effects of IFNbeta. Assays used to detect NAbs differ in their sensitivity and specificity, and there can be high variability between laboratories in how these assays are performed (e. g., types of cells, quantity of IFNbeta). This article reviews assays currently used for detecting NAbs to IFNbeta and discusses the development of an international standard NAb assay. The myxovirus resistance protein A (MxA) assay is recommended as the standard assay for the quantification of NAbs providing availability of reagents.

Abstract: BACKGROUND: The gene expression of the myxovirus-resistant protein A (MxA) gene is a sensitive measure of the biological response of therapeutically applied interferon-beta (IFNbeta) and of its reduced bioavailability due to inhibiting factors such as IFNbeta-induced neutralizing antibodies (NAbs). METHODS: We compared three methods for MxA mRNA quantification in 826 peripheral blood mononuclear cell (PBMC) samples obtained from patients with multiple sclerosis (MS). MxA mRNA measurements were performed using quantitative-competitive (qc)-PCR, real time-PCR, and the new semi-quantitative (sq)-PCR assay (MxA IBRIDOGEN). RESULTS: According to the treatment status (untreated samples versus NAb-negative treated samples), real time-PCR gave the highest specificity (93%). Slightly lower specificities were obtained with qc-PCR and sq-PCR (both 91%). qc-PCR showed the highest sensitivity (97%) compared with both real time-PCR (94%) and sq-PCR (95%). A positive correlation was found between qc-PCR and real time-PCR measurements (rspearman=0.776; p<0.0001), which also showed 90% agreement based on a statistically calculated threshold. Likewise, sq-PCR evaluations showed 84% and 79% agreement with qc-PCR and real time-PCR measurements, respectively. In addition, we showed a concordance of 89% between three sq-PCR kits. CONCLUSIONS: All three methods displayed high specificity for MxA gene expression analysis, allowing the detection of patients in whom IFNbeta did not have any biological action. qc-PCR and real time-PCR are both useful during clinical trials demanding quantitative data of biological activity, whereas sq-PCR could prove useful for routine screening purposes because it is easy to perform and can be done in not specialized laboratories.

Abstract: Glucocorticoid (GC)-induced osteoporosis is the leading form of secondary osteoporosis. Bone loss can be rapid. However, longitudinal studies at the very beginning of treatment are scarce. Patients relapsing from multiple sclerosis are treated with high-dose, short-term iv GCs. A number of them are young, without concomitant disease affecting bone and with no substantial impairment of mobility. Such patients were selected for the present study. Thirteen patients suffering from multiple sclerosis [11 females, two males; age 32 +/- 2 yr (mean +/- se)] and receiving iv methylprednisolone 15 mg/kg daily for 10 d completed the study. We measured serum osteocalcin (OC), aminoterminal propeptide of type I collagen (PINP), bone isoform of alkaline phosphatase (bALP), carboxyterminal telopeptide of type I collagen (CTX), and urinary calcium/creatinine ratio (uCa/Cr) during the 10-d cycle and 3 months later. Dual-energy x-ray absorptiometry and calcaneal quantitative ultrasonometry were performed before and 6 months after therapy. We found an immediate, impressive fall of OC and PINP (-80 +/- 3 and -54 +/- 5% at d 2, respectively), which persisted throughout the whole treatment period (P < 0.0001 for both markers). bALP levels showed only a modest decrease at d 6 (-19 +/- 7%, P < 0.05), with subsequent return to baseline in d 7-10. After 3 months, OC, PINP, and bALP levels rose to +51 +/- 22, +37 +/- 16 (not significant), and +61 +/- 17% (P < 0.01) with respect to baseline, respectively. uCa/Cr and CTX showed a progressive, marked increase during treatment, peaking at d 7-9 (+92 +/- 44 and +149 +/- 63%, respectively), with subsequent decrement at d 10 (P < 0.01 and P < 0.05, respectively) despite continuing GC administration. After 3 months, uCa/Cr and CTX levels were also higher than baseline. No change in quantitative ultrasonometry parameters and bone mineral density was observed 6 months after therapy. In conclusion, high-dose, short-term iv GC regimens cause an immediate and persistent decrease in bone formation and a rapid and transient increase of bone resorption. Our data also support the concept that discontinuation of such regimens is followed by a high bone turnover phase.

Abstract: BACKGROUND: MxA gene expression is one of the most appropriate markers of biological activity of exogenous interferon (IFN) beta. METHODS: We quantified MxA mRNA for five consecutive days in 62 patients treated with IFN beta (16, Avonex; 10, Betaferon; 24, Rebif 22; 12, Rebif 44), by quantitative-competitive polymerase chain reaction. Every three months, IFN beta induced neutralising antibodies (NAbs) were evaluated in sera using a cytopathic effect assay. RESULTS: Two categories of patients were identified: one group (49/62) had a sharp post-injection increase in MxA expression (defined as "IFN beta biological responder"), whereas the other group (13/62) had no MxA induction after IFN beta administrations (defined as "IFN beta biological non-responder"). In 11/13 biological non-responders, the persistent presence of NAbs correlated with abolished biological activity, independently of treatment regimen. The two remaining IFN beta biological non-responders were NAb-. Among the 49 IFN beta biological responders, biological activity was comparable between the four preparations on day 2 and 3 (+12 and +36 hours post-injection), but it was greater in Betaferon and both Rebif preparations on day 1, 4, and 5. In biological responders treated three times a week, only 82% (59/72) of injections were considered effective, compared with 100% (13/13) of Avonex injections. CONCLUSION: Our results suggest that an optimal IFN beta regimen is not yet available: Avonex, given once a week, shows lower cumulative biological activity. On the other hand, both Betaferon and Rebif, given three times a week, show 18% biologically ineffective injections and higher risk of developing NAbs, which abolish biological activity.

Abstract: A case of a severe necrotizing vasculopathic skin lesions occurred in a 43 year old women affected by multiple sclerosis (MS) submitted to IFNbeta-1b has been described. After two months of therapy the patient presented, in injection sites of the abdomen, arms and legs, numerous ulcers. A biopsy of the lesions was performed and evidenced confluent necrosis of the superficial and deep skin tissue with mild infiltration by inflammatory cells and thrombosis in deep blood vessels. The IFNbeta-1b was immediately discontinued and therapy with corticosteroids was started. After 12 months from the onset of the adverse reaction, the skin vasculopathic lesions cicatrised leaving sclerotic areas on the abdomen. Neutralizing antibodies against IFNbeta-1b (NABs) were strongly positive at the onset of the skin ulcers and slowly decreased until the recovery. A possible role of NABs in the development of the skin lesions has been considered.

Abstract: BACKGROUND: MxA is an antiviral protein exclusively induced by type I interferons (IFN) and some viruses, and MxA gene expression is one of the most appropriate markers for measuring the biologic activity of exogenous IFNbeta. METHODS: A new quantitative-competitive PCR method was used to quantify MxA mRNA in peripheral blood mononuclear cells of 99 treatment-naïve and 92 IFNbeta-treated patients with MS (22 Avonex, 17 Betaferon, and 53 Rebif-22). Every 3 months, IFNbeta-induced neutralizing antibodies (NAb) were evaluated in sera using a cytopathic effect assay. Three categories of patients were identified: NAb negative (NAb-), persistent NAb positive (NAb+, >or=2 consecutive positive samples), and isolated NAb+ (one positive sample). RESULTS: Treatment-naïve patients expressed detectable MxA mRNA levels (mean = 36 +/- 32 fg MxA/pg glyceraldehyde-3-phosphate dehydrogenase (GAPDH); range 1 to 160) and an upper normal threshold was established (mean + 3 SD = 132 fg MxA/pg GAPDH). IFNbeta-treated patients exhibited more than 11-fold higher levels (mean = 412 +/- 282 fg MxA/pg GAPDH; range 16 to 1,172). However, 17 patients did not exhibit an increase in MxA mRNA level; 15 of these 17 patients showed a concurrent Nab+ titer. Moreover, 13 were persistent NAb+. Isolated NAb+ patients did not show a decrease in bioavailability of IFNbeta (n = 9; mean = 567 +/- 366 fg MxA/pg GAPDH; range 83 to 1,120). In NAb- patients, bioavailability was comparable among the three different IFNbeta preparations 12 hours after injection. CONCLUSION: During IFNbeta therapy, the presence of NAb reduced or abolished bioavailability in a relevant percentage of patients. These data could be important for the early detection of patients with MS who are not responsive to IFNbeta therapy.

Abstract: Devic's neuromyelitis optica (NMO) is a clinical entity characterised by severe transverse myelitis, optic neuropathy and monophasic or recurrent course. We report the case of a woman affected by myelitis and optic neuritis suggesting Devic's disease. Diagnosis was supported by clinical, neuroradiological and biochemical findings. In 14 months, the patient developed 5 clinical exacerbations. Six cerebrospinal fluid (CSF) examinations were performed, 3 during relapses and 3 during remitting phases: all the CSF specimens obtained during relapses showed granulocyte pleocytosis and increased protein level, whereas CSF was normal during stationary phases. Oligoclonal banding was always absent. Spinal cord MRI showed altered signal at cervical and thoracic levels. We did not find any concomitant systemic disease. The case we report underlines the importance of CSF examination during clinical relapse in NMO diagnosis.

Abstract: Myxovirus A (MxA) is a protein that is specifically induced by treatment with type I cytokines and has proven to be a reliable biomarker of interferon-beta (IFNbeta) bioactivity. IFNbeta-induced MxA can be measured as either protein or mRNA in the blood of IFNbeta-treated patients with MS. In patients with MS who are treated with IFNbeta, loss of the MxA response is caused by high levels of anti-IFNbeta antibodies and is a sensitive marker of lost bioactivity.

Abstract: Previously, we have reported the development of a new quantitative-competitive polymerase chain reaction (qc-PCR) method to evaluate interferon-beta (IFNbeta) bioavailability in multiple sclerosis (MS) patients, by measuring mRNA of mixovirus resistance protein A (MxA). Here we show that our assay is also able to assess IFNalpha bioavailability in hepatitis C virus (HCV) patients treated with different IFNalpha regimens. Indeed, our method was able to detect a slight constitutive expression of MxA mRNA in untreated HCV patients (median=70 fgMxA/pgGAPDH) and a significant induction 12 h after the first IFNalpha administration (median=750 fgMxA/pgGAPDH).

Abstract: OBJECTIVE: To evaluate the incidence and the prevalence of neutralising antibodies (NABs) to three interferon beta (IFNbeta) products in patients with multiple sclerosis (MS). METHODS: Sera were tested from 125 patients with relapsing-remitting MS. Patients were treated with IFNbeta-1b (Betaferon, n = 29) 8 MIU subcutaneously every other day, IFNbeta-1a (Avonex, n = 44) 30 microg intramuscularly once weekly, or IFNbeta-1a (Rebif, n = 36) 22 microg subcutaneously three times weekly for 6 to 18 months. An additional 16 patients were treated with Rebif 22 microg intramuscularly once or twice weekly. NABs were assessed using the cytopathic effect assay before treatment and every three months during treatment. Patients with two or more consecutive positive samples were considered to be persistent NAB positive (NAB+). RESULTS: At baseline, no patients were NAB+. NABs developed during the first three months of treatment and continued to develop until month 18. Over 18 months of treatment, the risk of being persistent NAB+ was 31% for Betaferon, 15% for Rebif, and 2% for Avonex (Betaferon versus Avonex, p = 0.001; Betaferon versus Rebif, p = 0.19; Rebif versus Avonex, p = 0.04). In all patients with one or more NAB+ samples, the risk of becoming NAB+ was 38% for Betaferon, 18% for Rebif, and 7% for Avonex (Betaferon versus Avonex, p = 0.0007; Betaferon versus Rebif, p = 0.10; Rebif versus Avonex, p = 0.07). At month 18, the prevalence of persistent NAB+ patients was 31.6% for Betaferon, 18.7% for Rebif, and 4% for Avonex. Numbers of NAB+ patients observed were similar with intramuscular Rebif and with subcutaneous Rebif. CONCLUSION: The three IFNbeta preparations have different degrees of immunogenicity, with Betaferon producing the highest incidence of NABs and Avonex the lowest. These differences should be considered by neurologists when selecting treatment for their patients with MS because NABs can reduce both bioavailability and clinical efficacy of IFNbeta.

Abstract: BACKGROUND: Diffusion-tensor magnetic resonance imaging is sensitive to the more destructive aspects of multiple sclerosis (MS) evolution occurring outside and within T2-visible lesions and, as a consequence, holds promise for providing a more complete picture of primary progressive (PP) MS-related tissue damage than conventional magnetic resonance imaging. OBJECTIVE: To improve our understanding of PPMS by assessing the extent of occult pathological features in the normal-appearing white and gray matter of the brain using diffusion-tensor magnetic resonance imaging. METHODS: Ninety-six patients with PPMS, 47 patients with secondary progressive (SP) MS, and 44 healthy control subjects were studied. T2-hyperintense and T1-hypointense lesion volumes were calculated, and the volume of the whole brain tissue was measured. Diffusion-tensor magnetic resonance imaging scans were postprocessed and analyzed to obtain the mean diffusivity and fractional anisotropy histograms from the brain and from the normal-appearing white and gray matter in isolation. RESULTS: The mean T2-hyperintense and T1-hypointense lesion volumes were lower in patients with PPMS than in patients with SPMS, while the mean absolute brain volumes were similar in the 2 groups. The average lesion diffusivity was significantly higher in patients with SPMS than in patients with PPMS (P<.001). Histogram-derived metrics of the brain tissue and normal-appearing white and gray matter were significantly different between patients with PPMS and healthy subjects (range, P =.004 to <.001). Average diffusivity values were significantly higher in patients with SPMS than in patients with PPMS for all the tissues studied (range, P =.001 to <.001). Fractional anisotropy histogram-derived quantities did not significantly differ between the 2 patient groups (range, P =.94 to.03). CONCLUSION: This study confirms that, in patients with PPMS, normal-appearing white and gray matter are not spared by disease-related pathological processes, although they are affected to a lesser degree than in patients with SPMS.

Abstract: We analysed the role of dosage, route and frequency of administration of clinical grade interferon-beta (IFN-beta) preparations in inducing anti-IFN-beta antibodies (IFN-beta-Abs) in 5 groups of relapsing-remitting multiple sclerosis (RRMS) patients who were respectively treated as follows: 1) weekly intramuscular (i.m.) injections of 30 mg of recombinant IFN-beta1a (Avonex), 2) subcutis (s.c.) injections of 250 mg IFN-beta1b (Betaferon) every other day, 3) weekly i.m. injections of 250 mg IFN-beta1b (Betaferon), 4) s.c. injections of 22 mg of IFN-beta1a (Rebif) three times a week, and 5) i.m. injections of 22 mg of IFN-beta1a (Rebif) twice a week. IFN-beta-Abs were determined by ELISA. IFN-beta1b was more immunogenic than IFN-beta1a not only when administered s.c. every other day, but also when administered i.m. at a lower weekly dose; i.m. injection, however, significantly delayed the appearance, and induced lower serum levels of IFN-beta-Abs. In patients treated with s.c. IFN-beta1b, Ab levels peaked 3 to 9 months after therapy initiation, and then slowly, but progressively, declined to pre-therapy levels that in some patients were reached after three years. Patients treated with i.m. or s.c. IFN-beta1a only rarely developed IFN-beta-Abs, and then at very low titers. Overall, the i.m. weekly administration of IFN-beta1a was the less immunogenic treatment. In IFN-beta1b-treated patients, a wash-out period of two/three months was sufficient to bring the IFN-beta-Ab levels below the cut-off. Our findings suggest that the immunogenicity of IFN-beta1a is low, regardless of the route of administration and the dosage, while that of IFN-beta1b is high, and is significantly, but not completely reduced by i.m. administration. As IFN-beta-Abs are cross-reactive, a wash-out period is suggested when the preparation is changed from IFN-beta1b to IFN-beta1a in order to maintain the clinical benefits of the therapy.

Abstract: Intracellular expression of human myxovirus protein A (MxA) is exclusively induced by type I IFNs (IFNalpha,beta,omega) or by some viruses and it is strongly increased under IFN treatment. We set up an internally controlled quantitative-competitive polymerase chain reaction (qc-PCR) that quantifies MxA mRNA expressed in human peripheral blood mononuclear cells (PBMC). Our qc-PCR is accurate because the mean ratio of copy number estimated by qc-PCR to that quantified spectrophotometrically is 1.08+/-0.03, moreover it is repeatable with high sensitivity (1 fg MxA/pg GAPDH). MxA mRNA was tested in 47 Relapsing-Remitting Multiple Sclerosis (RR-MS) untreated patients and in 48 patients treated with one of the 3 IFNbeta licensed for MS (24 with Rebif, 14 with Avonex and 10 with Betaferon). All the 48 treated patients were negative to IFNbeta neutralising antibodies (NABs) as tested in our laboratory using a cytopathic assay (CPE). MxA mRNA levels were detectable in all untreated patients (mean 24+/-18 fg MxA/pg GAPDH) and significantly higher levels were found in all the treated patients 12 h after IFNbeta administration (mean 499+/-325 fg MxA/pg GAPDH); furthermore, the three types of IFNbeta showed comparable bioavailability. Our data indicate that the bioavailability of the three available types of IFNbeta can be evaluated by MxA qc-PCR.

Abstract: In patients with primary progressive (PP) multiple sclerosis, brain MRI lesion activity and burden are low, despite the presence of severe neurological impairment. On the contrary, the degree of cord atrophy and diffuse tissue damage in the brain and cervical cord have been found to be associated with clinical disability. Against this background, this study aimed at providing an in vivo indirect assessment of brain and cervical cord pathology in a large cohort of PP multiple sclerosis patients, using conventional MRI and magnetization transfer imaging (MTI). Ninety-one PP multiple sclerosis patients, 36 secondary progressive (SP) multiple sclerosis patients and 30 healthy controls underwent brain and cervical cord MRI scans, using dual echo (brain) or fast short-tau inversion recovery (cervical cord) MTI and T(1)-weighted sequences. For the brain, T(2) hyperintense and T(1) hypointense lesion volumes were calculated and the volume of the whole of the brain tissue measured. For the cervical cord, the number and burden of lesions and the cross-sectional area were assessed. MTI scans were post-processed and analysed to obtain magnetization transfer ratio (MTR) histograms from the whole of the brain and cervical cord tissue and from the normal-appearing brain tissue in isolation. In PP multiple sclerosis patients, brain, normal-appearing brain tissue and cervical cord MTR histogram-derived metrics revealed the presence of diffuse tissue damage whose characteristics did not significantly differ from those of SP multiple sclerosis patients, even though SP multiple sclerosis patients had higher MRI-visible lesion burdens. None of the correlations between MRI or MTI measures obtained from the brain and the cord were significant. PP multiple sclerosis patients' disability was significantly, albeit weakly associated with a composite MR model including measures of loss and intrinsic damage of cervical cord tissue. Our data indicate the presence of a diffuse tissue damage undetectable by conventional MRI in PP multiple sclerosis patients, whose extent seems to match that of SP multiple sclerosis patients with similar levels of disability. They also suggest that the severity of multiple sclerosis pathology in the cervical cord is one of the factors contributing to neurological impairment in PP multiple sclerosis.

Abstract: The presence and titer of neutralizing antibodies (NABs) was evaluated by an antiviral biological assay in 387 samples of 111 multiple sclerosis (MS) patients treated with one of the three commercial preparations of interferon beta (IFNbeta). Fifty NAB positive samples were found in 19 patients: 11 treated with IFNbeta-1b (Betaferon(R)) and eight with IFNbeta-1a (five with Avonex(R) and three with Rebif(R)). All the 38 NABs+ samples of patients treated with IFNbeta-1b cross-reacted with IFNbeta-1a of both commercial types. The median level of neutralizing units (NUs) of the sera was higher when tested against IFNbeta-1a than against IFNbeta-1b (p=0.000 vs. Avonexr(R) and p=0.003 vs. Rebif(R)).In line with these data, the NABs+ sera of patients treated with IFNbeta-1a cross-reacted with IFNbeta-1b and the level of NUs were lower when tested against IFNbeta-1b than against IFNbeta-1a (p=0.003). The different amount of NUs against IFNbeta types 1a and 1b could be due to the presence of aggregates in the IFNbeta-1b preparation. The different levels of cross-reactivity of NABs could reduce the bioavailability and therapeutic efficacy of IFNbeta in NABs+ patients switching from IFNbeta-1b to IFNbeta-1a.

Abstract: Eight relapsing-remitting multiple sclerosis (MS) patients were tested for the level of transforming growth factor beta1 (TGFbeta1) mRNA in peripheral blood mononuclear cells every 15 days for 6 months. Disease activity was evaluated every 4 weeks by magnetic resonance imaging (MRI) and neurological examination. An inverse correlation was found between the level of TGFbeta1 mRNA and MRI disease activity. The level of TGFbeta1 mRNA predicted the presence of disease activity in the scans performed 2-4 weeks later with high sensitivity (88%) and specificity (87.5%) suggesting that TGFbeta1 mRNA quantification could be an indicator of disease activity in MS.

Abstract: Quantification of tumor necrosis factor-alpha (TNF-alpha) mRNA in peripheral blood mononuclear cells (PBMC) could provide information about disease activity in multiple sclerosis (MS); however, specific competitive methods must be utilized. A competitor cDNA, having the same sequence of the target TNF-alpha cDNA, a part from an internal 49-bp deletion, was generated and used to set-up a quantitative polymerase chain reaction (PCR) to quantify mRNA of TNF-alpha. Competitor and target were co-amplified using the same primers. The rates of generation of competitor and target TNF-alpha conformed closely to the prediction of the mathematical model, and a high level of accuracy and reproducibility was achieved. The method was applied to quantify TNF-alpha mRNA in PBMC of normal subjects and multiple sclerosis (MS) patients both during clinical relapses and remissions. A statistically significant higher level of TNF-alpha mRNA was detected during relapses than during remissions. High levels of TNF-alpha mRNA were found in 44% of relapses and 12% of samples during remissions, suggesting that TNF-alpha mRNA synthesis is abnormal in MS.

Abstract: Microglia expressing keratan sulfate (KS) was studied in normal central nervous system (CNS) and in rat neonatal brain cultures. The majority of KS+ cells are ramified microglia located in the brain parenchyma; positive cells were only exceptionally found in extraparenchymal structures. KS+ cells are ubiquitous, but their density is heterogeneous throughout the CNS. Serial sections incubated with anti-KS MAb and MAb against the complement receptor type 3 (CR3) revealed a higher number of CR3+ cells and double immunofluorescence showed the presence of two microglial populations: the first expressing both KS and CR3, the second expressing only CR3. Two sets of microglial cells were found also in neonatal rat microglial cultures where only a low percentage of microglial cells expressing CR3 was also KS+. KS was not induced by microglia activation.

Abstract: Pig endocardiosis is a pathological process affecting cardiac valves that is characterised by the accumulation of glycosaminoglycans (GAG) in the extracellular matrix. To investigate the involvement of GAG in the condition, the morphology of the mitral valves from 23 affected pigs and seven normal controls was studied and qualitative and quantitative biochemical analyses of GAG were made. Gross and histopathological lesions were characterised by valve enlargement, collagen disorganisation and myxoid degeneration. No differences between normal and diseased valves were detected by lectin histochemistry. Electron microscopy revealed myofibroblast differentiation of many fibroblasts. A statistically significant increase of total GAG and hyaluronan was detected in the mitral valves of the pigs with endocardiosis by spectrophotometric, electrophoretic and densitometric analysis of the extracted GAG. Although it is not known whether the change in hyaluronan is a primary event or a result of other changes in the extracellular matrix, its accumulation in association with myofibroblast differentiation suggests that it plays a pathogenetic role in pig endocardiosis.

Abstract: Subsets of neurons ensheathed by perineuronal nets containing chondroitin unsulfated proteoglycan have been immunohistochemically mapped throughout the rat central nervous system from the olfactory bulb to the spinal cord. A variable proportion of neurons were outlined by immunoreactivity for the monoclonal antibody (Mab 1B5), but only after chondroitinase ABC digestion. In forebrain cortical structures the only immunoreactive nets were around interneurons; in contrast, throughout the brainstem and spinal cord a large proportion of projection neurons were surrounded by intense immunoreactivity. Immunoreactivity was ordinarily found in the neuropil between neurons surrounded by an immunopositive net. By contrast, within the pyriform cortex the neuropil of the plexiform layer was intensely immunoreactive even though no perineuronal net could be found. The presence of perineuronal nets could not be correlated with any single class of neurons; however a few functionally related groups (e.g., motor and motor-related structures: motor neurons both in the spinal cord and in the efferent somatic nuclei of the brainstem, deep cerebellar nuclei, vestibular nuclei; red nucleus, reticular formation; central auditory pathway: ventral cochlear nucleus, trapezoid body, superior olive, nucleus of the lateral lemniscus, inferior colliculus, medial geniculate body) were the main components of the neuronal subpopulation displaying chondroitin unsulfated proteoglycans in the surrounding extracellular matrix. The immunodecorated neurons found in the present study and those shown by different monoclonal antibodies or by lectin cytochemistry, revealed consistent overlapping of their distribution patterns.

Abstract: Anti-chondroitin unsulfated proteoglycan (COS-PG) 1B5 monoclonal antibody (MAb) and Vicia villosa agglutinin (VVA) recognize the same neuronal subset. Moreover, when in double immunofluorescence study the sections were digested with chondroitinase ABC (ChABC), a procedure necessary to create the epitope of 1B5 MAb, and incubated with VVA, no VVA positive neurons were detected. This finding suggests that VVA binds terminal N-acetyl-galactosamine present in the glycidic chains of COS-PG or of glycoproteins that form perineuronal molecular aggregates with COS-PG.

Abstract: Recently we reported that the keratan sulphate epitope recognised by the monoclonal antibody 5D4 is expressed by a population of ramified microglia in adult rats. As ramified microglia is believed to differentiate from ameboid microglia during postnatal development, we studied the rat brain from birth to 90 postnatal days of life with the monoclonal antibody 5D4. Contrary to all the other microglia markers until now described, keratan sulphate is not expressed by ameboid microglia and by macrophages but appears on the surface of microglia only when the cells are differentiated and show ramified processes. The keratan sulphate positive cells become evident at different times in different central nervous system areas; the first were localised in the pyriform cortex and brainstem from the end of the second postnatal week. These observations suggest that keratan sulphate expression on microglia cells is induced by differentiation and by a resting functional state. Moreover the 5D4 monoclonal antibody showed a strong diffuse positive staining of some cortical, thalamic and white matter areas during the first two postnatal weeks. This staining was transient and it does not seem biologically correlated with the expression of the keratan sulphate on differentiated microglia.

Abstract: A 20-year-old patient was born with epidermolysis bullosa and a severe, slowly progressive muscle disease. Skin biopsy demonstrated junctional epidermolysis bullosa. Muscle biopsy demonstrated degenerative changes with increase in connective tissue, fibre size variability, rods and cytoplasmic bodies, central nuclei. In muscle biopsy dystrophin, chondroitin unsulphate, chondroitin 4-sulphate, chondroitin 6-sulphate, heparan sulphate, collagen III, collagen IV and VI, laminin, and fibronectin were normally distributed. This is the first report of the association of a form of congenital muscular dystrophy with junctional epidermolysis bullosa and, together with the previous reports of muscle involvement in epidermolysis bullosa simplex and dystrophica, it suggests the existence of a syndrome characterized by the contemporaneous presence of skin and muscle involvement.

Abstract: The c-MET proto-oncogene encodes the tyrosine kinase receptor for hepatocyte growth factor (HGF), also known as scatter factor, a powerful mitogen and motility factor for epithelial cells. We now show that the two previously described forms of the Met/HGF receptor, the intact p190MET and the truncated p140MET, are expressed in physiological conditions in the human central nervous system (CNS). The receptors were identified by Western blot analysis with monoclonal antibodies directed against different epitopes. By immunohistochemical staining the Met/HGF receptor was found to be expressed in a homogeneous cell population, equally distributed between the grey and the white matter, showing morphological features and immunochemical markers specific for the resident microglial cells. These data suggest a possible role for the c-MET proto-oncogene and HGF in microglial reactions to CNS injuries.

Abstract: We used six monoclonal antibodies (MAb) recognizing epitopes within keratan sulfate (KS) chains for an immunocytochemical study of adult rat brain. One of the MAb selectively stained microglia and their ramified processes. KS-positive cells were found throughout the CNS in both paraffin-embedded and cryostat sections; the greatest number were present in hippocampus and brainstem. In the cortex the positive processes of some cells surrounded neuronal somata. In the white matter the processes were both parallel and perpendicular to the axon bundles. Double staining showed that KS-positive cells did not express astrocytic or oligodendroglial markers. By immunoelectron microscopy, the positivity was localized around the perikarya and cell processes of small cells with peripheral chromatin clumps and dark cytoplasm, which often contained secondary lysosomes. The KS-positive cells did not contribute to myelin sheaths and were not surrounded by a basal membrane. In addition to the cellular staining, three other MAb stained the white matter diffusely. Anti-KS MAb are therefore proposed as immunohistochemical markers for ramified microglia in both paraffin and cryostat sections of adult rat brain.

Abstract: Chondroitin sulfate proteoglycan (CS-PG) bearing glycosaminoglycan (GAG) chains containing unsulfate (COS) and 6-sulfate (C6S) disaccharides was immunolocalized in rat and human CNS by using monoclonal antibodies (MAb) specific for the two disaccharides. The immunostaining with both MAb was restricted to the periphery of a neuronal subset in rat and human CNS. Double immunofluorescence showed codistribution of the antigens around the same neuronal population. The staining with anti-COS MAb was stronger than with anti-C6S MAb, suggesting that the proteoglycan (PG) contains mainly COS disaccharides. In different rat cortical areas, 40-60/mm2 positive interneurons were found, the visual cortex showing the highest value. In human cortex, positivity was also observed around the soma of some pyramidal cells. In the rat, positive neurons were also localized in deep cerebellar nuclei, reticular nucleus of the thalamus, and other structures of the midbrain and hindbrain. CA3 region of hippocampus and the external layer of pyriform cortex were characterized by positivity of the neuropil. Immunoelectronmicroscopy showed the antigens in the extracellular space around the neuronal soma, the synaptic elements and the cell processes of the neuropil. The neuronal surface of the soma and of the proximal dendrites were positive, but the pre- and postsynaptic membranes and clefts were negative.

Abstract: In this paper we report that the integrin complex alpha 1/beta 1, a laminin/collagen receptor, is expressed on cultured foreskin microvascular endothelium, but is absent on endothelial cells from large vessels such as the aorta and umbilical and femoral veins. The restricted expression of integrin alpha 1/beta 1 to microvascular endothelium was also demonstrated in vivo, by immunohistochemical staining of human tissue sections. Alpha 1 specific antibodies reacted strongly with endothelial cells of small blood vessels and capillaries in several tissues, but not with endothelium of vein and arteries of umbilical cord. Expression of integrin alpha 1 can be induced in cultured umbilical vein endothelial cells by treatment with 5 ng/ml tumor necrosis factor alpha (TNF alpha). Induction of alpha 1 subunit expression also occurred after treatment of umbilical vein endothelium with 10(-5) M retinoic acid or with 10 nM PMA; Maximal induction of alpha 1 integrin was reached after 48 h of treatment and costimulation with TNF alpha and PMA resulted in a synergistic effect. The induction of alpha 1 integrin changed the adhesive properties of umbilical vein endothelial cells, by increasing the adhesiveness to collagen, laminin, and laminin fragment P1, while adhesion to fibronectin and laminin fragment E8 remained constant. The alpha 1 integrin is thus a marker of a specific population of endothelial cells and its expression confers distinctive properties of interaction with the underlying basal membrane.

Abstract: We have studied the extracellular matrix composition of cultured glial cells by immunocytochemistry with different monoclonal and polyclonal antibodies. Double immunofluorescence experiments and metabolic labeling with [3H]glucosamine performed in different types of cerebellar and cortical cultures showed that bipotential progenitors for type-2 astrocytes and for oligodendrocytes (recognized by the monoclonal antibody LB1 at early stages of their development) synthesize chondroitin sulfate (CS) and deposit this proteoglycan in their extracellular matrix. The distribution of the various [3H]glucosamine-labeled glycosaminoglycans between the intracellular and the extracellular space was different. CS was present both within the cells and in the culture medium, although in different amounts. Bi-potential progenitors became also O4-positive during their development in vitro. At the stage of O4-positivity they were still stained with antibodies against CS. However, when the progenitor cells were maintained in serum-free medium and differentiated into Gal-C-positive oligodendrocytes, they became CS-negative. In the presence of fetal calf serum in the culture medium, the bipotential progenitors differentiated into GFAP-positive type-2 astrocytes. These cells still expressed CS: their Golgi area and their surface were stained with anti-CS antibodies. Staining with monoclonal antibodies specific for different types of CS (4-sulfate, 6-sulfate, and unsulfated) revealed that both bipotential progenitors and type-2 astrocytes synthesized only chondroitin 4-sulfate. Type-1 astrocytes were negative for both the polyclonal and the monoclonal anti-CS antibodies. Finally, type-2 astrocytes and their progenitors were weakly stained with anti-laminin antibodies and unstained with anti-fibronectin. Type-1 astrocytes were positive for both anti-laminin and anti-fibronectin antibodies and appeared to secrete fibronectin in the extracellular space.

Abstract: Chondroitin 4-sulfate proteoglycan (C4S-PG) was localized both in rat and human central nervous system (CNS) by monoclonal and polyclonal antisera recognizing the 4-sulfate disaccharide (C4S). In the rat the whole CNS was studied in serial coronal sections. A positive extracellular meshwork was observed both in white and grey matters. In the white matter (WM) C4S-PG formed a network around myelinated axons, sparing myelin sheaths and axoplasms. The neuropil of the grey matter (GM) showed a positive meshwork constituted by delicate intermingling filaments. The cytoplasms of neuronal, glial and endothelial cells were negative. Stronger straining than in the neuropil was observed around the soma and the proximal part of the cell processes of some neurons located in the cortex, in the deep cerebellar nuclei and in some other CNS nuclei. A similar pattern was also observed in human CNS, the only difference being a smaller amount of cortical neurons surrounded by a rim of C4S-PG. This study shows that a PG bearing C4S disaccharide is located extracellularly in the rodent and human CNS and that C4S disaccharides can be present in different types of CNS proteoglycans (PGs).

Abstract: The localization and quantitation of glycosaminoglycans classes (GAGs) were studied in human meningiomas. Meningiomas presented high amounts of these compounds and electrophoretic separation revealed that they were 90% sulphated. The Alcian method and a polyclonal antiserum against chondroitin sulphate were used to localize the different GAGs in tissue sections. Quantitative and qualitative differences and different tissue distributions of GAGs were observed among transitional, syncytial and fibroblastic meningiomas. Syncytial meningiomas presented the lowest amount of GAGs and the immuno- and histochemical studies showed that they were located only in vessels and connectival trabeculae. Transitional meningiomas contained the highest concentration of GAGs; the percentage of the different GAG classes was similar to that observed in the syncytial oncotype indicating a quantitative but not qualitative difference between the two oncotypes. The high amount of GAGs in transitional meningiomas was attribute to the whorls, the structures stained by the histochemical and immunohistochemical techniques. The tumoral parenchyma of these two oncotypes was negative. On the contrary, fibroblastic meningiomas showed a fine meshwork among tumoral cells containing chondroitin sulphate and heparan sulphate. Biochemical data were consistent with the histochemical and immunohistochemical findings revealing a high percentage of chondroitin sulphate and heparan sulphate in fibroblastic meningiomas. This study suggests that the three meningioma types have different abilities to produce extracellular matrix components.

Abstract: Leukocyte glutamate dehydrogenase (GDH) was studied in 29 patients affected by progressive cerebellar ataxia (PCA) and in 20 healthy controls. Eight GDH-deficient patients, with GDH activity 2 SD below mean value of controls, were identified. GDH deficiency did not identify a subgroup of PCA by characteristic pattern of inheritance and/or age of onset of disease. However, the GDH-deficient patients presented more neurological signs than non-GDH-deficient patients. A significant correlation was observed between GDH deficiency and the presence of extrapyramidal signs, supranuclear palsy, absence of osteotendineal reflexes and neurogenic electromyographical findings.

Abstract: Chondroitin, chondroitin 6-sulphate, chondroitin 4-sulphate and dermatan sulphate proteoglycans were immunolocalized by monoclonal antibodies applied to human muscle sections digested with chondroitinase. In normal muscle the 4 proteoglycans presented a different extracellular localization: unsulphated chondroitin sulphate (chondroitin) was present in the endomysium and around capillaries, chondroitin 6-sulphate in the basal membrane zone, chondroitin 4-sulphate in the vessel adventitia, in the endomysium around capillaries and, to a lesser degree, in the perimysium, dermatan sulphate in the perimysium and, to a lesser extent, in the vessel adventitia and in the endomysium around capillaries. The enlarged endomysium of pathological muscle contained chondroitin and chondroitin 4-sulphate. Chondroitin 6-sulphate and dermatan sulphate did not seem present in the increased connective tissue. No peculiar pattern was observed in the various neuromuscular diseases studied. The specific extracellular distribution, the different biochemical composition and the different ability to bind to other extracellular components suggest a different biological role of these compounds. Chondroitin 6-sulphate is a component of a highly specialized extracellular structure, namely basal membrane. Chondroitin and chondroitin 4-sulphate participate in the composition of actively changing extracellular matrix such as the endomysium in pathological muscle. On the other hand, dermatan sulphate is a constituent of the perimysium that is a more static extracellular structure.

Abstract: We have used an antibody raised against the bovine nasal cartilage proteoglycan chondroitin sulfate (CS) digested with chondroitinase ABC (anti-CS serum) to stain cerebellar glial cells maintained in culture. In cultures grown in the presence of serum, the antibody stained a subclass of GFAP+ astrocytes which we have previously shown to selectively bind the monoclonal antibodies A2B5 and LB1. Also the direct bipotential precursors of these cells, capable of differentiating into GFAP+ astrocytes or into Gal-C+, O1+ oligodendrocytes depending on the culture conditions, were stained, but stopped to produce CS when they differentiated into oligodendrocytes.

Abstract: A polyclonal rabbit antiserum was utilized to localize chondroitin sulfate in human gliomas. Tissue sections were digested with chondroitinase ABC to create the antigenic determinant on the chondroitin sulfate proteoglycan molecule. Normal CNS tissue showed a positive immunohistochemical staining both in white and gray matter, sparing the cytoplasm of glial and neuronal cells. Differentiated astrocytomas presented the same pattern as the normal CNS. Anaplastic astrocytomas and glioblastomas showed progressive reduction of parenchymal positivity as anaplasia increased. These data suggest that chondroitin sulfate is a character expressed by differentiated CNS cells and that it is lost with dedifferentiation. Vascular structures presented positive material in the adventitia in all the oncotypes. A discontinuous positivity was observed in the basal membrane zone of the vessels.

Abstract: Glycosaminoglycans (GAGs) were isolated, separated by electrophoresis and quantified in 36 neurosurgical specimens of human gliomas and in 8 samples of normal white and gray matter. Gliomas of various degrees of malignancy exhibited different GAG patterns. Total GAG concentration was three times higher in low grade gliomas than in normal white matter. The mean percentage of single GAG classes was usually similar in both tissues, although in certain tumor samples a higher percentage of hyaluronate was found. GAG patterns in anaplastic astrocytomas, however, more closely resembled normal white and gray matter, both quantitatively and qualitatively. Glioblastomas, on the other hand, showed high GAG concentrations, in particular of heparan sulfate and dermatan sulfate. This finding could be secondary to the abundant vessels and mesodermal material associated with this oncotype. The hyaluronate/sulfated GAGs ratio was lower in oligodendrogliomas than in low grade astrocytomas. This biochemical feature may be correlated with the alcianophilia found in the honey-comb degeneration of oligodendrogliomas. The significance of these findings as they relate to tumor histology and biology have been discussed.

Abstract: The immunohistological localization of chondroitin sulfate (CS) has been studied in normal and pathological human muscle. The bovine nasal cartilage proteoglycan digested with chondroitinase ABC (BNC-PG-Ch ABC) has been utilized for the production of a rabbit polyclonal antiserum. In vitro studies showed that the antiserum binds to the unsaturated disaccharide that remains attached to the core protein after digestion of the CS chains with chondroitinase ABC (Ch ABC). As the disaccharide is created specifically by Ch ABC digestion of the CS chains, the antiserum allows the immunolocalization of CS on tissue sections digested with Ch ABC. The immunohistochemical study on normal and pathological muscle demonstrated a localization of CS in all the extracellular structures: endomysium, perimysium, muscle spindle capsule and intrafusal space. In pathological conditions, the CS was raised in all the cases with increased connective tissue, showing a pattern comparable to that obtained with fibronectin and collagen III. None of the pathological conditions displayed any peculiar character of CS distribution. This finding does not support a primary role for CS in the pathogenesis of muscular dystrophy.

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Abstract: The effects of collagenase on the immunohistochemical demonstrability of laminin, fibronectin and Factor VIII/RAg in human nervous tissue have been studied. The influence of this, and other proteolytic enzymes such as pepsin and trypsin, has been investigated in relation to different fixatives. Collagenase gave better results with Carnoy fixed material than after formalin fixation; unlike trypsin and pepsin, it did not produce tissue digestion.

Abstract: The Doppler technique has only been used in neurological practice to evidence local vessel pathology such as occlusion or stenosis. Both common carotid artery and internal jugular vein flows can change not only because of pathological processes of the vessels but also because of impedance of their distribution territory. In this report we analyze the relationship between flow velocity, one of the parameters of blood flow, and intracranial impedance variations which occur in cerebral concussion, tumors and acute vascular cerebral pathology. During our observations we noticed that the diastolic wave of the velocity curve of the common carotid artery is a very important signal of the flow variations in the internal carotid artery and, in turn, of variations in cerebral flow. We studied the behaviour of the common carotid artery velocity curve in our patients both during clinical disease development and during the action of mannitol in the acute phases of the disease. We found that the ultrasonic patterns during antiedema action were similar to the ones obtained during the recovery period. We were able to note some differences and some similarities of the curve morphology in relation to generalized or focal causes of cerebral edema. This may be very important considering that at present no non-invasive and therefore repeatable technique is available for monitoring cerebral blood flow in intracranial hypertension.

Abstract: The occurrence, distribution and concentration of GAGs in ENU and MNU experimental brain tumors induced in the rat are reported. GAGs have been histochemically studied by Alcian Blue methods; they have been quantified and qualitatively evaluated by electrophoresis of brain extracts. The pattern of GAGs in normal rats is consistent with the data of the literature. No GAG accumulation precedes the tumor development. Early neoplastic proliferations, oligodendroglial and mixed glial microtumors are strongly alcian-positive; the alcianophilia spares clusters of cells developing a cytoplasm. In large tumors, GAGs are histochemically demonstrable in the honey-comb areas of oligodendrogliomas and in peripheral infiltration areas of polymorphic gliomas. The role of the normal nervous tissue and oligodendroglial cells in the accumulation of the GAGs is discussed. The accumulated GAGs seem to rise from the nervous tissue included in the tumors, rather than from the metabolism of tumor cells.

Abstract: The availability of antisera against collagen and non-collagen proteins of the extracellular matrix has opened new possibilities of studying fibrous infiltration in muscular diseases. We have examined muscle biopsies from 5 controls and 27 patients with various neuromuscular diseases by immunofluorescence and peroxidase-anti-peroxidase. We investigated the distribution of fibronectin and of laminin, a protein present in basement membranes. In normal muscle both were present around blood vessels, axons, muscle spindles and muscle fibres. In addition fibronectin filled the endomysial and perimysial space, the endoneurium and the space between the intrafusal fibres. In pathological muscle laminin distribution was similar to that in normal muscle, but some atrophic fibres appeared to have a thickened contour and irregular profiles were occasionally observed in the absence of histologically demonstrable muscle fibres. Fibronectin was increased in all the conditions with thickened endomysium and perimysium, without displaying any disease-specific character. Findings are compared with the few published observations on fibronectin and collagen types.

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Abstract: The occurrence and the distribution of GAGs have been studied histochemically in 224 human cerebral tumors by means of Alcian blue techniques. In the normal peritumoral gray matter the alcianophilia is stronger than in the white matter and demonstrated the presence of HA and CS. In the glioma group the alcianophilia, due to HA and CS, is mainly related to the presence of infiltrated cortex. In the other tumors, GAGs are histochemically disclosed in relation to collagen, reticulin, mesodermic areas, etc. The vessels of every tumor show a positive staining for HA, CS and HS. The histochemical findings are consistent with the biochemical ones as reported in Part I, even though the significance of GAGs in cerebral tumors remains unknown.

Abstract: The case of a 57-year-old woman is described with a two months history of proximal muscle weakness and pain, marked hypotrophy and brisk reflexes. Clinical investigation demonstrated normal serum CK, myopathic EMG and osteomalacia. Muscle biopsy showed type II fibre atrophy and mitochondrial alterations without inclusions. Further examinations including a jejunal biopsy revealed malabsorption accounting for osteomalacia. At autopsy diffuse nodular lipomatosis of the small bowel was detected (Acta neurol. belg., 1982, 82, 65-71).

Abstract: In order to define the significance of minimal histological and ultrastructural abnormalities in Duchenne carriers, 18 normal healthy volunteers were examined by muscle biopsy. Light microscopy evidenced occasional internal nuclei and less frequent small round and angular fibres. Variability of fibre size, increase in connective tissue, necroses and basophilia found in carriers were not present in controls. Histograms constructed on ATPase stain sections demonstrated variability in distribution of fibre types both in normals and in carriers. Fibre size was more variable in carriers, where a significant decrease of the size of type II B fibres was observed. Electron microscopy evidenced knots in overcontraction, myofibrillary widening, subsarcolemmal accumulation of mitochondria and Z band streaming, which have been reported also in carriers. The results are discussed and compared with the data of the literature.

Abstract: An epidemiological investigation on Duchenne muscular dystrophy in Turin and its province has been carried out for the period 1955-974. The incidence of the disease proved to be 24 X 10(-5), and the prevalence 2.15 X 10(-5). The mutation rate was 80 X 10(-6). The data are compared with the literature. Segregation analysis on many families was performed in order to find the theoretical number of carrier among Duchenne mothers. The importance of epidemiology for genetic counselling is stressed.

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Abstract: The presence of myoglobin in the urine is a symptom of rhabdomyolysis occurring in many muscular affections. Muscle tissue from seven patients who had presented one of more episodes of myoglobinuria has been studied by histological, histochemical, and biochemical methods. Three cases belonging to the same family and an additional one revealed a muscular CPT deficiency. Two of those cases were females. In two other cases a toxic etiology was suggested, one due to heroin and the other to associated fenfluramine and phenformin. In the last case the origin of rhabdomyolysis was not clearly defined and viral infection, drug intolerance or electrolytic imbalance were proposed. The cases are discussed in the light of the literature and the possible etiopathogenetic mechanisms are reviewed.

Abstract: 5 cases of late motor neuron degeneration following poliomyelitis are presented. CPK, CSF, EMG and spine radiology were studied. In all cases, muscle biopsy evidenced neurogenic alterations both in previously affected limbs and in newly affected ones. Mitochondrial abnormalities were found in 1 case. The findings are discussed in the light of the literature with particular regard to the pathogenesis of the disease. The case with abnormal mitochondria is stressed for the possible relationship of this pathology with systemic degenerative diseases.

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Abstract: A case of late motor neuron degeneration following poliomyelitis with abnormal mitochondria in muscle fibers is presented with two additional cases of systemic neurogenic muscular atrophy (Charcot-Marie-Tooth disease). Muscle biopsy revealed a neurogenic pattern of variable severity in all cases. Subsarcolemmal zones of hyperactivity and hyperpositive intermyofibrillar collections of granular material present in a variable proportion of type I fibers were demonstrated by oxidative enzymes. Ultrastructurally they corresponded to abnormal mitochondria, with paracrystalline inclusions in one case. The finding is discussed in the light of the previous literature on mitochondrial myopathies. Mitochondrial alterations are not specific and their significance in neurogenic conditions is debated.

Abstract: A case of centronuclear myopathy is presented. The presence of central nuclei in almost all fibres, the existence of type I fibres only, the histochemical pattern of a negative central zone with a perinuclear halo and a hyperactive rim with oxidative enzymes and the ultrastructural data are discussed in the light of the previous literature. The possible relationships with other myopathies are taken into consideration as well as the fact that central nuclei may be a non-specific change in several conditions. Consequently centronuclear myopathy could turn out to be a syndrome from which different entities can be isolated.

Abstract: A case of late onset ophthalmoplegia and dysphagia is presented. Serum enzymes, ECG, EEG, thyroid function and edrophonium test were normal. Muscle biopsy revealed the presence at oxidative enzymes of 5% of fibres displaying subsarcolemmal hyperactivity and a coarse network pattern, mainly involving type II fibres. At electron microscopy mitochondrial abnormalities with paracrystalline inclusions were detected. The case is discussed in the light of the heterogeneity of ocular myopathies and their overlapping both with one another and with the so called mitochondrial myopathies.

Abstract: A case of acute muscle necrosis with probable myoglobinuria is presented. The patient had been taking excessive doses of phenformin and fenfluramine, and a toxic etiology is suggested. Muscle necrosis is tentatively attributed either to the combined effect of these drugs on some underlying biochemical abnormality of muscle or to a defect of oxygen utilization secondary to myoglobin alterations.

Abstract: The two-dimensional immunoelectrophoresis on cellulose acetate, already utilized for serum proteins, has been applied to CSF proteins. The technical modifications which allow the use of unconcentrated CSF are described. By utilizing a particularly suitable supporting medium, a good separation of proteins is achieved; consequently narrow-based peaks are obtained and the isoantigens can be displayed as distinct components in spite of closely similar migration velocities. The preliminary results of this method are given. Particulary interesting appears a beta-migrating protein and the gamma-migrating oligoclonal bands. The first may present an anodic cleavage suggesting that it may be the beta1C/beta1A-globulin. If this hypothesis is confirmed, the relative concentrations of the two components can be investigated. No clear-cut peaks corresponding to the gamma-migrating oligoclonal bands have been observed, their place being taken by a low precipitation line continuous with the IgG peak. The reasons for this phenomenon may be tentatively ascribed to a local specificity of these IgG.