Efficacy of repeated intrathecal triamcinolone acetonide application in progressive multiple sclerosis patients with spinal symptoms

Background

There are controversial results on the efficacy of the abandoned, intrathecal predominant methylprednisolone application in multiple sclerosis (MS) in contrast to the proven effectiveness in intractable postherpetic neuralgia.

Methods

We performed an analysis of the efficacy of the application of 40 mg of the sustained release steroid triamcinolone acetonide (TCA). We intrathecally injected in sterile saline dissolved TCA six times within three weeks on a regular basis every third day in 161 hospitalized primary and predominant secondary progressive MS patients with spinal symptoms. The MS patients did not experience an acute onset of exacerbation or recent distinct increased progression of symptoms. We simultaneously scored the MS patients with the EDSS and the Barthel index, estimated the walking distance and measured somatosensory evoked potentials. Additionally the MS patients received a standardized rehabilitation treatment.

Results

EDSS score and Barthel index improved, walking distance increased, latencies of somatosensory evoked potentials of the median and tibial nerves shortened in all MS patients with serial evaluation (p < 0.0001 for all variables). Side effects were rare, five patients stopped TCA application due to onset of a post lumbar puncture syndrome.

Conclusions

Repeated intrathecal TCA application improves spinal symptoms, walking distance and SSEP latencies in progressive MS patients in this uncontrolled study. Future trials should evaluate the long-term benefit of this invasive treatment.

     

Expansion and functional relevance of high-avidity myelin-specific CD4+ T cells in multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease in which myelin-specific T cells are believed to play a crucial pathogenic role. Nevertheless, so far it has been extremely difficult to demonstrate differences in T cell reactivity to myelin Ag between MS patients and controls. We believe that by using unphysiologically high Ag concentrations previous studies have missed a highly relevant aspect of autoimmune responses, i.e., T cells recognizing Ag with high functional avidity. Therefore, we focused on the characterization of high-avidity myelin-specific CD4+ T cells in a large cohort of MS patients and controls that was matched demographically and with respect to expression of MHC class II alleles. We demonstrated that their frequency is significantly higher in MS patients while the numbers of control T cells specific for influenza hemagglutinin are virtually identical between the two cohorts; that high-avidity T cells are enriched for previously in vivo-activated cells and are significantly skewed toward a proinflammatory phenotype. Moreover, the immunodominant epitopes that were most discriminatory between MS patients and controls differed from those described previously and were clearly biased toward epitopes with lower predicted binding affinities to HLA-DR molecules, pointing at the importance of thymic selection for the generation of the autoimmune T cell repertoire. Correlations between selected immunological parameters and magnetic resonance imaging markers indicate that the specificity and function of these cells influences phenotypic disease expression. These data have important implications for autoimmunity research and should be considered in the development of Ag-specific therapies in MS.     

The role of antibodies in multiple sclerosis

B cells, plasma cells, and antibodies are commonly found in active central nervous system (CNS) lesions in patients with multiple sclerosis (MS). B cells isolated from CNS lesions as well as from the cerebrospinal fluid (CSF) show signs of clonal expansion and hypermutation, suggesting their local activation. Plasma blasts and plasma cells maturating from these B cells were recently identified to contribute to the development of oligoclonal antibodies produced within the CSF, which remain a diagnostic hallmark finding in MS. Within the CNS, antibody deposition is associated with complement activation and demyelination, indicating antigen recognition-associated effector function. While some studies indeed implied a disease-intrinsic and possibly pathogenic role of antibodies directed against components of the myelin sheath, no unequivocal results on a decisive target antigen within the CNS persisted to date. The notion of a pathogenic role for antibodies in MS is nevertheless empirically supported by the clinical benefit of plasma exchange in patients with histologic signs of antibody deposition within the CNS. Further, such evidence derives from the animal model of MS, experimental autoimmune encephalomyelitis (EAE). In transgenic mice endogenously producing myelin-specific antibodies, EAE severity was substantially increased accompanied by enhanced CNS demyelination. Further, genetic engineering in mice adding T cells that recognize the same myelin antigen resulted in spontaneous EAE development, indicating that the coexistence of myelin-specific B cells, T cells, and antibodies was sufficient to trigger CNS autoimmune disease. In conclusion, various pathological, clinical, immunological, and experimental findings collectively indicate a pathogenic role of antibodies in MS, whereas several conceptual challenges, above all uncovering potential target antigens of the antibody response within the CNS, remain to be overcome.     

Multiplexing analysis of the polyspecific intrathecal immune response in multiple sclerosis

Intrathecal synthesis of the antibodies specific to neurotrofic viruses: measles (M), rubella (R), Varicella-Zoster (Z), and/or H. simplex (H), known as "MRZH-reaction" plays important diagnostic role in multiple sclerosis (MS). Whereas the analysis of the oligoclonal IgG bands provides high sensitivity, the MRZH-reaction shows high specificity, and hence these methods complement each other. For the first time we applied multiplexing bead-based technology to simultaneously analyze cerebrospinal fluid (CSF) and serum concentrations of antibodies against these viruses, and to calculate the antibody specific indices (ASI's). The method shows reasonable precision: intra-assay, 2.9-6.7%, and inter-assay, 2.0-3.2%. The results are comparable with these obtained with other methods (ELISAs), including two runs of the certified external quality control schemes. Eighty-one percent of the MS cases (n=27) and none of the sex- and age-matched controls (n=14), except one subject with "borderline" anti-measles ASI of 1.5, showed intrathecal synthesis of IgG against at least one of the viruses discussed. The ratios of the MRZH-positive cases in the MS group were: 12/22 for M, 12/19 for R, 13/26 for Z, and 7/26 for H. We conclude that the multiplexing technology can be applied as a tool to study the intrathecal immune response in the diagnosis of MS.     

Orienting reaction in patients with multiple sclerosis

A polygraphic study of the somatic (EMG), autonomic (finger plethysmogram, galvanic skin reaction, respiration, pulse, and EEG (acoustic-evoked potential and EEG-blocking reaction) components of the orienting reaction elicited by an auditory stimulus was performed in 71 patients with multiple sclerosis and in 74 matched normal subjects (control group). The study showed a significantly less intense orienting reaction in patients with multiple sclerosis than in control normal subjects. The severity of this responsiveness disturbance depended on the patients' age at symptom onset, major symptom types on admission, and the relapse frequency. The orienting reaction changes found in patients with multiple sclerosis may be ascribed to the diffuse demyelinating lesions in the nervous system of these patients, which generate, apart from the so polymorphous clinical manifestations of this disease, also important responsiveness disturbances.     

Oxidative stress in patients with multiple sclerosis

It is well known that brain and nervous system cells are prone to oxidative damage because of their relatively low content of antioxidants, especially enzymatic ones, and of the high levels of both membrane polyunsaturated fatty acids (PUFA) and iron easily released from injured cells. We have investigated the oxidative stress in the blood (plasma, erythrocytes and lymphocytes) of 28 patients affected with multiple sclerosis (MS) and of 30 healthy age matched controls, by performing a multiparameter analysis of non-enzymatic and enzymatic antioxidants--Vitamin E (Vit. E), ubiquinone (UBI), reduced and oxidized glutathione (GSH, GS-SG), superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and fatty acid patterns of phospholipids (PL-FA). PL-FA and Vit. E were assayed by GC-MS; UBI and GSH/GS-SG by HPLC; SOD, GPX and CAT by spectrophotometry. In comparison to controls, patients with MS showed significantly reduced levels of plasma UBI (0.21 +/- 0.10 vs. 0.78 +/- 0.08 mg/ml, p < 0.001), plasma Vit. E (7.4 +/- 2.1 vs. 11.4 +/- 1.8 mg/ml, p < 0.01), lymphocyte UBI (8.1 +/- 4.0 vs. 30.3 +/- 7.2 ng/ml blood, p < 0.001) and erythrocyte GPX (22.6 +/- 5.7 vs. 36.3 +/- 6.4 U/g Hb, p < 0.001). This blood antioxidant deficiency was associated with plasma levels of PL-PUFA--especially C20:3 n-6 and C20:4 n-6--significantly higher than controls. In conclusion, the blood of patients with MS shows the signs of a significant oxidative stress. The possibility of counteracting it by antioxidant administration plus an appropriate diet, might represent a promising way of inhibiting the progression of the disease. Antioxidant supplements should include not only GSH repleting agents, but also Vit. E, ubiquinol, and selenium.     

Increased serum CNTF level in patients with amyotrophic lateral sclerosis

The role of ciliary neurotrophic factor (CNTF) in the etiopathogenesis of amyotrophic lateral sclerosis (ALS) is still not clear. The aim of this study was to measure serum CNTF level in patients with ALS. The study involved 36 ALS patients and 43 control group subjects. CNTF was measured by the enzyme-linked immunosorbent assay. Results showed that the serum CNTF level in whole group of ALS patients was significantly higher from those in the total control group subjects. CNTF level was not dependent on the clinical state of the ALS patients, type of ALS onset, or duration of the disease. Results indicate that CNTF may be a reactive component resulting from the disease, but it cannot be a marker of ALS activity. It is possible that the increase in serum CNTF level is the result of the muscle denervation seen in this disease.     

Serial immunoprecipitation assays for interferon--(IFN)-beta antibodies in multiple sclerosis patients

We devised a sensitive, radioimmunoprecipitation assay (RIPA) for anti-interferon (IFN)-beta-binding antibody (BAB) detection. Our RIPA showed good agreement with a reference RIPA (mean difference, -3.2 +/- 10.6 AU), and detected BAB to both IFN-beta-1a and IFN-beta-1b. Neutralizing antibodies to IFN-b (NAB) were also determined with a standard method. BAB and NAB were measured in 393 serum samples from 77 multiple sclerosis (MS) patients treated with IFN-beta-1a or -1b, who were studied over two years, and subsequently classified as responders and non-responders. BAB were found at higher concentrations, and more frequently detected, in IFN-beta-1b- than in IFN-beta-1a-treated patients, and, at highest titres, preferentially in patients who were positive for NAB. However, in our series of MS patients, both titres and frequency of detection of BAB or NAB did not differ between IFN-b responders and non-responders.     

VH4 gene segments dominate the intrathecal humoral immune response in multiple sclerosis

A characteristic feature of the CNS inflammatory response in multiple sclerosis (MS) is the intrathecal synthesis of IgG and the presence of oligoclonal bands. A strong correlation between CD138(+) plasma blast numbers in MS cerebrospinal fluid (CeSF) and intrathecal IgG synthesis suggests that these cells are the major Ab-secreting cell type in MS CeSF. Sequencing of V regions from CD138(+) cells in MS CeSF has revealed somatically mutated and expanded IgG clonotypes consistent with an Ag-targeted response. In the present study, single-cell RT-PCR analysis of CD138(+) cells from 11 MS patients representing differing clinical courses and stages of disease identified expansion of CD138(+) cells with functionally rearranged V(H)4 gene segments as an overriding feature of MS CeSF repertoires. V(H)4 dominance was attributed to the preferential selection of specific V(H)4 genes, particularly gene segment V(H)4-39, which displayed a significant enrichment in CeSF compared with MS peripheral blood B cells. A modest increase in V(H)4 prevalence among MS peripheral blood IgG memory cells was also noted, suggesting that factors shaping the CD138 repertoire in CeSF might also influence the peripheral IgG memory cell pool. These results indicate a highly restricted B cell response in MS. Identifying the targets of CeSF plasma cells may yield insights into disease pathogenesis.     

Increased sister-chromatid exchange rate and its regression during prolonged incubation in lymphocyte cultures from patients with multiple sclerosis

Peripheral venous blood lymphocytes from multiple sclerosis patients, cultured for 72 h in the presence of phytohemagglutinin, appeared to have a higher sister-chromatid exchange (SCE) rate than cells from matched controls. Prolongation of the incubation time to 9 days by adding interleukin-2 to the cultures, caused the cells from the MS patients to lose their increased SCE frequency, so that the mean rate no longer differed from that of the controls. The SCE rate of the controls did not change significantly on prolonged incubation.     

Increased frequencies of myelin oligodendrocyte glycoprotein/MHC class II-binding CD4 cells in patients with multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease characterized by infiltration of pathogenic immune cells in the CNS resulting in destruction of the myelin sheath and surrounding axons. We and others have previously measured the frequency of human myelin-reactive T cells in peripheral blood. Using T cell cloning techniques, a modest increase in the frequency of myelin-reactive T cells in patients as compared with control subjects was observed. In this study, we investigated whether myelin oligodendrocyte glycoprotein (MOG)-specific T cells could be detected and their frequency was measured using DRB1*0401/MOG(97-109(107E-S)) tetramers in MS subjects and healthy controls expressing HLA class II DRB1*0401. We defined the optimal culture conditions for expansion of MOG-reactive T cells upon MOG peptide stimulation of PMBCs. MOG(97-109)-reactive CD4(+) T cells, isolated with DRB1*0401/MOG(97-109) tetramers, and after a short-term culture of PMBCs with MOG(97-109) peptides, were detected more frequently from patients with MS as compared with healthy controls. T cell clones from single cell cloning of DRB1*0401/MOG(97-109(107E-S)) tetramer(+) cells confirmed that these T cell clones were responsive to both the native and the substituted MOG peptide. These data indicate that autoantigen-specific T cells can be detected and enumerated from the blood of subjects using class II tetramers, and the frequency of MOG(97-109)-reactive T cells is greater in patients with MS as compared with healthy controls.     

A novel assay to detect low-titred antibodies to interferon beta in multiple sclerosis patients

Neutralizing antibodies (NAbs) may compromise interferon (IFN) clinical efficacy in patients with multiple sclerosis (MS) receiving IFN-beta treatment. When bioassays are used for anti-IFN-beta antibody detection, they are unable to discriminate between NAbs or other interfering substances with anti-IFN activity. Here we report the development of an anti-IFN-beta Western blot method that facilitates the detection of IFN low-titred antibodies and characterizes such low neutralizing activity as specifically due to the presence of particular IFN antibodies. The assay was characterized using serum samples from patients with MS treated with IFN-beta. It was developed by adding anti-IFN-positive antibody sera to Dynabeads M-280 tosylactivated followed by Western blot analysis. All sera samples from MS patients with IFN-betala NAbs (< or = 50 t1/10) proved to be antibody-positive using this new method and, more importantly, four of 27 binding antibody-negative sera samples were scored as IFN antibody-positive. The method was found to be rapid, specific and sensitive and consistent with respect to well-established antiviral neutralization or commercial enzyme-linked immunosorbent assays.     

VIP in cerebrospinal fluid of patients with multiple sclerosis

The concentration of VIP was measured radioimmunochemically in cerebrospinal fluid (CSF) from 14 healthy volunteers and from 22 patients with multiple sclerosis. Significantly lower levels of VIP was obtained in the patients (18 +/- 3 pmol/l) than in controls (37 +/- 4 pmol/l). There was no correlation between the level of VIP in CSF and other CSF parameters such as albumin. IgG or cell content; nor between VIP concentration and the physical handicap or neuropsychiatric symptoms. There was a trend towards lower values of VIP in patients with steadily progressing rather than intermittent course of the disease but the difference between the groups was not significant.     

Longitudinal assessment of antisaccades in patients with multiple sclerosis

We have previously demonstrated that assessment of antisaccades (AS) provides not only measures of motor function in multiple sclerosis (MS), but measures of cognitive control processes in particular, attention and working memory. This study sought to demonstrate the potential for AS measures to sensitively reflect change in functional status in MS. Twenty-four patients with relapsing-remitting MS and 12 age-matched controls were evaluated longitudinally using an AS saccade task. Compared to control subjects, a number of saccade parameters changed significantly over a two year period for MS patients. These included saccade error rates, latencies, and accuracy measures. Further, for MS patients, correlations were retained between OM measures and scores on the PASAT, which is considered the reference task for the cognitive evaluation of MS patients. Notably, EDSS scores for these patients did not change significantly over this period. These results demonstrate that OM measures may reflect disease evolution in MS, in the absence of clinically evident changes as measured using conventional techniques. With replication, these markers could ultimately be developed into a cost-effective, non-invasive, and well tolerated assessment tool to assist in confirming progression early in the disease process, and in measuring and predicting response to therapy.     

Increased level of fibrinogen chains in the proteome of blood platelets in secondary progressive multiple sclerosis patients

Epidemiological studies indicate a high risk of stroke, heart failure and myocardial infarction in patients with multiple sclerosis, especially in its secondary progressive (SPMS) phase. Some ischaemic events are directly associated with abnormal platelet functions and their prothrombotic activity. Recent reports, including this study, confirm the increased activation of circulating platelets in SPMS, and also show increased platelet reactivity, among other responses, as well as strong aggregation. In this current study, we conducted a comparative analysis of the platelet proteome in SPMS patients and in healthy controls, to demonstrate the quantitative and qualitative differences likely to affect functional changes observed in SPMS. During densitometry evaluation of 2‐D fluorescence difference gel electrophoresis, we observed differences between the electrophoretic patterns of SPMS platelets and the control samples. To determine a detailed characterisation of the proteome changes in the SPMS patients’ blood platelets, in the next stage, we performed mass spectrometry of selected spots and indicated the increased presence of four proteins (fibrinogen, α‐2 macroglobulin, septin‐14 and tubulin β‐1 chain). The most important of these is the increased amount of prothrombotic protein, fibrinogen, which seems to confirm the accuracy of the imaging and potentially explains the increased risk of platelet‐origin thrombotic events. This study provides new knowledge of the potential existence of the molecular mechanisms responsible for the acceleration of the platelet pro‐coagulant function in SPMS. This can help to identify new targets for therapy, which can then be used not only in the second stage of the disease.