Reduced adhesion of PBMNCs to endothelium in methylprednisolone-treated MS patients: preliminary results

Methylprednisolone (MP) is a synthetic steroid commonly used in the treatment of multiple sclerosis (MS) relapses. It has a wide spectrum of activities on immune cells: it might also act by preventing mononuclear cell/endothelium adhesion. We studied adhesion phenomena between cultured human umbilical vein endothelial cells (HUVECs) and PBMNCs (CD45⁺, CD14⁺) from 6 MS patients treated in vivo with MP. We also studied fluctuations in CD11a and CD18 levels on lymphocytes and monocytes, as well as changes in serum sICAM-1 and sVCAM-1 concentrations. After MP treatment, PBMNCs adhesion to endothelium decreased at 3 h, while it went back to baseline levels at 24 h. A tendency to increase in both CD11a and CD18 on the surface of lymphocytes was detected, while an increase in serum sVCAM-1 was seen at 3 h.     

Imbalances in circulating lymphocyte subsets in Hu antibody associated paraneoplastic neurological syndromes

In paraneoplastic neurological syndromes (PNS) associated with small cell lung cancer (SCLC) and Hu antibodies, neuron-specific Hu antigens expressed by the tumour hypothetically trigger an immune response that cross-reacts with Hu antigens in the nervous system, resulting in tumour suppression and neuronal damage. To gain more insight into the hypothesized cell-mediated immune pathogenesis of these syndromes, we analysed the circulating lymphocyte subsets in untreated patients with SCLC, PNS and Hu antibodies ( n  = 18), SCLC without PNS ( n  = 19) and controls ( n  = 29) using flow cytometry. SCLC patients with PNS had a variety of imbalances within their circulating lymphocyte subsets as compared with SCLC patients without PNS and healthy controls: (i) a lymphopenia of the major subsets (i.e. B, CD4⁺ and CD8⁺ T lymphocytes); (ii) increased proportions of activated CD4⁺ and CD8⁺ T cells; (iii) reduced numbers of terminally differentiated effector CD8⁺ T cells and cells with a cytotoxic T-cell phenotype (CD56⁺ and CD57⁺). Although indirect, our data provide further support for the involvement of T cells in the pathogenesis of Hu antibody associated PNS.     

CD4 and CD8 lymphocyte subsets in cerebrospinal fluid and peripheral blood from patients with multiple sclerosis, meningitis and normal controls

Objectives - To study the distribution of CD4⁺ and CD8⁺ T-cell subsets in cerebrospinal fluid (CSF) and peripheral blood from patients with multiple sclerosis (MS), meningitis, other neurological diseases and healthy controls.
Material and methods - The expression of markers for naive and memory cells (CD45RA⁺ and CD45RO⁺), and helper/inducer cells (CD29⁺) on CD4⁺ cells as well as CD45RO⁺ and killer/effector (S6F1⁺) on CD8⁺ cells was investigated in cerebrospinal fluid (CSF) and peripheral blood from patients with multiple sclerosis (n=28), meningitis (n = 13), other neurological diseases (n = 16), and healthy controls (n = 16) by 2-color flow cytometry.
Results - The majority of T cells in the CSF of the 4 groups exhibited the phenotype of memory cells (CD45RO⁺) on both CD4⁺ and CD8⁺ cells. The proportion of helper/inducer (CD29TD4⁺ in CD4⁺) cells was also larger in the CSF compared to peripheral blood in the 3 patient groups and controls investigated. In contrast, CD8⁺ cells with killer/effector (S6F1+) phenotype were fewer in CSF compared to peripheral blood in all 4 groups. There were no significant differences between patients and controls regarding the distribution of these activation markers in the CSF or peripheral blood.
Conclusion - Our observations support the notion that activated T cells of both CD4⁺ and CD8⁺ phenotype selectively pass the blood-brain barrier under both pathological and normal conditions.     

Immunophenotyping in Tourette syndrome – a pilot study

Background and purpose:  The cause of Tourette syndrome (TS) is not precisely known, although several lines of evidence point at an involvement of the immune system in its pathogenesis.
Results:  Here, we report the results of a pilot study investigating frequently analysed lymphocyte surface markers in 20 adult patients with TS (16 males; 37.3 ± 15.8 years) and 20 matched controls (16 males; 37.5 ± 15.3 years). Statistical analysis revealed significant differences for the investigated lymphocyte surface markers. The difference in CD69⁺/CD22⁺-B cells (23.0 ± 10.5% vs. 13.1 ± 6.1%; P  = 0.001) and in CD95⁺/CD4⁺-T cells (41.5 ± 12.1% vs. 24.6 ± 10.0%; P  = 0.0001) was still significant after Bonferroni–Holm correction.
Conclusion:  Our preliminary data indicate that TS may be associated with an increased peripheral immune activity.     

A substantial number of Rasmussen syndrome patients have increased IgG, CD4+ T cells, TNFα, and Granzyme B in CSF

Purpose:   We studied the immunologic molecules in cerebrospinal fluid (CSF) and discussed their evolutional changes in pediatric patients with Rasmussen syndrome (RS).
Methods:   CSF samples collected from 27 patients with RS (average onset age, 7.5 ± 5.6 years) were studied. Cell count, protein, glucose, albumin, chloride, and immunoglobulin G (IgG) levels were measured by conventional methods. Surface markers of lymphocytes in CSF were examined by a cell sorter. Granzyme B, interferon γ (IFNγ), interleukin 4 (IL-4), tumor necrosis factor α (TNFα), and IL-12 in CSF were quantitated by enzyme-linked immunosorbent assay (ELISA). Autoantibodies against GluR ε2 (NR2B) were examined by immunoblot.
Results:   The data of the first CSF examination showed that IgG levels (Mann-Whitney U test, p < 0.01), CD4⁺ T cells (p = 0.02), TNFα levels (p < 0.01), and Granzyme B levels (p < 0.01) were elevated compared with disease controls. White blood cell count, IFNγ level, IL-12 level, and Granzyme B level were elevated, especially in the early stage of disease. CD4⁺ T cells, CD8⁺ cells, CD3⁺ T cells, IgG levels, and TNFα levels were elevated at all stages of disease evolution. Protein levels and albumin levels were elevated in the progressed stage. Autoantibodies against GluR ε2 (NR2B) (IgG) were found in 50% of patients in the early stage, and the positive rate was low at the progressed stage.
Discussion:   The present findings suggest that complex pathophysiologic mechanisms involving CD4⁺ T cells and CD8⁺ T cells change evolutionally during the progression of RS. A crucial cytotoxic process occurs in the early stage, and declines in the progressed stage.     

Parkinson's disease and immunological abnormalities: increase of HLA-DR expression on monocytes in cerebrospinal fluid and of CD45RO+ T cells in peripheral blood

The etiology of Parkinson's disease is mainly unknown. Immune abnormalities have been described, but the cause of such abnormalities has not been resolved. We examined by two-colour flow cytometry HLA-DR antigen expression on monocytes from cerebrospinal fluid (CSF) and blood and, moreover, lymphocyte subpopulations (CD4⁺ CD45RO⁺, CD4⁺ CD45RA⁺, CD8⁺ CD11b^+high) in peripheral blood from patients with Parkinson's disease compared with age-matched patients with other neurological diseases (OND) and tension headache. We found higher HLA-DR expression on CSF monocytes compared with blood monocytes. This difference was restricted to Parkinson's disease patients. T helper cell analysis revealed a decreased percentage of CD45RA⁺"naive" and an increased percentage of CD45RO⁺"memory" T cell subset from CD4⁺ T cells in peripheral blood of patients with Parkinson's disease compared with patients with tension headache. The proportions of CD8⁺ CD11b^+high"suppressor" T cells remained unchanged, among the three patient groups compared. A selective loss of CD4⁺ CD45RA⁺ cells, previously observed in diseases like multiple sclerosis and Down's syndrome as compared with healthy controls suggests a common immunological abnormality in neurological disorders.     

Effect of chronic Li+ treatment on free intracellular Mg2+ in human neuroblastoma SH-SY5Y cells

Objectives:  Previous findings have demonstrated Li⁺/Mg²⁺ competition at therapeutic intracellular Li⁺ levels after acute Li⁺ treatment in human neuroblastoma SH-SY5Y cells. In the current study, we examined whether Li⁺/Mg²⁺ competition exists at therapeutically relevant extra- and intracellular [Li⁺] after chronic Li⁺ loading times.
Methods:  In human neuroblastoma cells, intracellular free Mg²⁺ was determined by fluorescence spectroscopy with the fluorophore furaptra. Intracellular Li⁺ and Mg²⁺ were measured by atomic absorption spectrophotometry.
Results:  After loading of the neuroblastoma cells with 1–2 mM extracellular Li⁺ for 24–72 h, the observed, increased intracellular free [Mg²⁺] levels were significantly higher (p < 0.03) than those in matched Li⁺ free cells, and intracellular [Li⁺] was found to be at therapeutic intracellular levels (0.7–1.5 mM).
Conclusions:  The results demonstrate that Li⁺/Mg²⁺ competition exists after chronic treatment with Li⁺ at therapeutically relevant intracellular Li⁺ levels in neuroblastoma cells. We found differences between acute and chronic Li⁺ treatment effects on the extent of Li⁺/Mg²⁺ competition. Possible reasons for these differences are discussed.     

Treatment of a patient with myasthenia gravis using antibodies against CD25

Objectives –  To measure clinical and immunological parameters in a patient with myasthenia gravis (MG) treated with antibodies against CD25 (basiliximab, Simulect^®).
Patient and methods –  Injections of basiliximab were given repeatedly together with cyclosporin A and corticosteroids for 9 months to a patient with severe MG. Her muscle function score was monitored and the immunological parameters were followed using ELISA, flow cytometry and radioimmunoassay.
Results –  The patient improved moderately and corticosteroid treatment could be withdrawn. The percentage of activated CD4⁺ T cells decreased during treatment, while that of 'naïve' T cells increased. The serum levels of sCD28, sCD152, sCD80, sCD86 and IL-10 decreased. The treatment was stopped due to repeated infections.
Conclusion –  Treatment with basiliximab appears to be suitable only for severely ill patients who do not respond to conventional treatments. However, careful monitoring of side effects is necessary.     

Analysis of lymphocyte subpopulations in cerebrospinal fluid and peripheral blood in patients with multiple sclerosis and inflammatory diseases of the nervous system

We analysed different subsets of lymphocytes from peripheral blood (PB) and cerebrospinal fluid (CSF) by flow cytometry in order to determinate alterations in patients with multiple sclerosis (MS) in acute relapse and viral inflammatory neurological disease (IND). We found increased levels of adhesion molecules (LFA-1 and β₁ integrin) in the CSF of patients with MS and IND compared to NIND. CD4 ⁺/CD8 ⁺ ratio was significantly higher in CSF of MS as compared with all groups analysed and compared with PB. We detected a significantly higher expression of the interleukin-2 receptor in PB of MS patients when compared with other groups. In patients with IND a significant higher expression of the interleukin-2 receptor was found in the CSF compared with MS and NIND. Our findings indicate that the activation of T lymphocytes primarily occurs in the peripheral immune compartment in MS and the increase of adhesion molecules in CSF is related to inflammatory disorders and not only to MS.     

V region T cell receptor repertoire in Parkinson's disease

Introduction - Restricted usage of Vα and β genes has been found in several diseases, which exert autoreactive T cells. So far no information on T cell receptor (TCR) usage in degenerative diseases is available. Since T cells may be involved in pathogenesis of Parkinson's disease, the analysis of the TCR repertoire is of importance. Material and methods - We have tested the frequency of 6 Vβ-subtypes and the most common Vα-subfamily on peripheral blood lymphocytes in 21 PD patients and 20 controls, separately on CD4⁺ and CD8⁺ T cells. For our study Diversi - T™, the first available Mab set specific for TCR V regions, was used. Results -As a results, no significant differences were found with one exception, i.e., lower frequency of Vβ8(a) expression on CD8⁺ T cells in PD patients than in controls. Conclusion - The failure of preferential usage or lack of usage of the tested V-chain alleles by CD4⁺ T cells argues against an involvement of helper T cells in PD induction.     

Pediatric State of the Art Symposium: Seizures and Epilepsy Complicating Medical Conditions in Childhood 7:30 p.m.–9:30 p.m.

¹ Dennis J. Dlugos , ² Mary L. Zupanc , ¹ Robert R. Clancy , ³ Anna Janss , and ⁴ Benjamin Eidelman (   ¹ Division of Neurology, The Children's Hospital of Philadelphia, Philadelphia, PA ;   ² Department of Neurology, Children's Hospital of Wisconsin, Milwaukee, WI ;   ³ Department of Neurology, Emory University School of Medicine, Atlanta, GA ; and   ⁴ Department of Neurology, Mayo Clinic, Jacksonville, FL )
This symposium will address advances in clinical management and translational research regarding acute provoked seizures and chronic epilepsy in children with complex medical conditions, such as congenital heart disease, oncological disorders, and organ transplantation. These medical conditions are all complicated by acute provoked seizures and chronic epilepsy, involve treatment with medications with complex interactions with anti-epileptic drugs, and provide opportunities for translational research.
The goals of the symposium are:

• 1)  

  Understand the etiology, pathophysiology and treatment of acute provoked seizures in children with congenital heart disease, CNS and non-CNS neoplasms, and organ transplantation.

       
  
  

Impairment of circulating CD4+CD25+ regulatory T cells in patients with chronic inflammatory demyelinating polyradiculoneuropathy

Abstract   Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an immune-mediated peripheral nervous system disease. CD4⁺CD25⁺ T regulatory cells (Tregs) have been unequivocally shown to be critical in maintaining immune tolerance and preventing auto-immune diseases by suppressing self-reactive T cells. Thus, we hypothesized that the numbers and/or the function of Tregs would be deranged during the progressive or relapse phases of CIDP. The number of Tregs was determined by flow cytometry according to their characteristic CD4⁺CD25^high membrane phenotype. Functional characterization of Tregs was analyzed by suppression of proliferation and secretion of cytokines by co-cultured effector CD4⁺CD25⁻ T cells. FOXP3 message expression level was assessed by quantitative real-time polymerase chain reaction. The results showed significant reduction in both the number and the suppressive function of Tregs in the patients with CIDP compared with healthy controls. Also, Tregs isolated from CIDP patients expressed lower levels of FoxP3 mRNA. During the progressive or the relapsing phases of CIDP, the number of Tregs was reduced, and the suppressive function of them decreased. These findings may be helpful to our understanding of the possible role of Tregs in the pathogenesis of CIDP.     

Clinical Epilepsy: Adult

¹ Ahmed T. Abdelmoity , and ¹ Amit Verma (   ¹ Peter Kellaway Section of Neurophysiology, Department of Neurology, Baylor College of Medicine, Houston, TX )
Rationale: Benzodiazepines are rarely used as long term therapy for patients with epilepsy. However, once traditional antiepileptic drugs have failed an adjunct trial may be justified to help reduce seizure frequency.  

     

14.

IGF-I promotes neuronal migration and positioning in the olfactory bulb and the exit of neuroblasts from the subventricular zone     

Anahí Hurtado-Chong  María J. Yusta-Boyo  Eva Vergaño-Vera  Alessandro Bulfone  Flora de Pablo  Carlos Vicario-Abejón 《The European journal of neuroscience》2009,30(5):742-755

While insulin-like growth factor-I (IGF-I) supports neuronal and glial differentiation in the CNS, it is largely unknown whether IGF-I also influences neuronal migration and positioning. We show here that the pattern of olfactory bulb (OB) layering is altered in Igf-I ^−/− mice. In these animals, Tbr1⁺-glutamatergic neurons are misplaced in the mitral cell layer (ML) and the external plexiform layer (EPL). In addition, there are fewer interneurons in the glomerular layer and the EPL of the Igf-I ^−/− mice, and fewer newborn neurons are incorporated into the OB from the forebrain subventricular zone (SVZ). Indeed, neuroblasts accumulate in the postnatal/adult SVZ of Igf-I ^−/− mice. Significantly, the positioning of Tbr1⁺-cells in a primitive ML is stimulated by IGF-I in cultured embryonic OB slices, an effect that is partially repressed by the phosphoinositide 3-kinase (PI3K) inhibitor. In OB cell cultures, IGF-I increases the phosphorylation of disabled1 (P-Dab1), an adaptor protein that is a target of Src family kinases (SFK) in the reelin signalling pathway, whereas reduced P-Dab1 levels were found in Igf-I ^−/− mice. Neuroblast migration from the rostral migratory stream (RMS) explants of postnatal Igf-I ^−/− was similar to that from Igf-I ^+/+ explants. However, cell migration was significantly enhanced by IGF-I added to the explants, an effect that was repressed by PI3K and SFK inhibitors. These findings suggest that IGF-I promotes neuronal positioning in the OB and support a role for IGF-I in stimulating neuroblast exit from the SVZ into the RMS, thereby promoting the incorporation of newly formed neurons into the OB.     

15.

T cell subsets in multiple sclerosis: a serial study     

M. Calopa  J. Bas  M. Mestre  T. Arbizu  J. Peres  E. Buendia 《Acta neurologica Scandinavica》1995,92(5):361-368

The relevance of abnormalities in the distribution of peripheral blood T lymphocyte subsets to the clinical manifestations of multiple sclerosis is not firmly established. A clinical and immunological follow-up of relapsing-remitting multiple sclerosis patients was performed in order to study the relationship of immune changes with the clinical course of the disease. Twenty patients were monitored monthly during a mean time of nine months for peripheral blood lymphocyte subsets (CD3, CD4, CD8, CD19), including the immunoregulatory subsets CD4CD29 (helper-inducer), and CD4CD45RA (suppressor-inducer) and activated T helper cells (CD4CD25) by flow cytometry. A total of 14 untreated relapses was included. The most significant observations were a decrease in T suppressor-inducer CD4⁺ CD45RA⁺ subset during clinical relapses ( P = 0.028) that was also detectable one month before ( P = 0.020) and the lack of changes in CD4⁺ CD29⁺ and CD8⁺ T cells. In addition, variations in the percentage of CD4⁺CD25⁺ activated T helper cells were not associated with clinical exacerbations. These results indicate the existence of a temporal association of immune changes in peripheral blood, but not activation, with the clinical manifestations of multiple sclerosis.     

16.

Expression of chemokine receptors on peripheral blood mononuclear cells of patients with immune-mediated neuropathies treated with intravenous immunoglobulins     

C. Trebst  K. Brunhorn  M. Lindner  A. Windhagen  M. Stangel 《European journal of neurology》2006,13(12):1359-1363

Intravenous immunoglobulin (IVIg) is an efficacious treatment for immune-mediated neuropathies like Guillain–Barré syndrome (GBS), chronic inflammatory demyelinating neuropathy (CIDP), and multifocal motor neuropathy (MMN). In the pathogenesis of immune-mediated neuropathies chemokines and their receptors play a crucial role. Using flow cytometry we examined whether IVIg modulates chemokine expression repertoires of T cells and monocytes. The expression of inflammatory chemokine receptors CCR1, CCR2, CCR4, CCR5, CCR6 and CXCR3 was investigated on circulating T-cell subsets, and CCR1, CCR2 and CCR5 on circulating monocytes before and after IVIg treatment in patients with immune-mediated neuropathies (MMN, n  = 7; GBS, n  = 1; CIDP, n  = 2). Furthermore, the homing potential of T cells was analyzed by the expression of CCR7, a chemokine receptor known to be utilized by mature T cells to recirculate into secondary lymphoid organs. In contrast to studies in chronic heart failure, no differences in expression patterns before and after IVIg treatment of any of the investigated chemokine receptors were found. Furthermore, the proportion of CD45RO-positive CD4⁺ or CD8⁺ T-cell subsets was not changed by IVIg treatment. Thus, we concluded that modulation of the expression of chemokine receptors on circulating leukocytes by IVIg is not a mode of action in immune-mediated neuropathies.     

17.

Tuesday July 4, 200612:00–13:30Hall 1Platform SessionDrug Therapy II     

《Epilepsia》2006,47(S3):12-14

¹ P. Williamson , ¹ C. Tudur Smith , ² J. Sander , and ¹ A. Marson (   ¹ University of Liverpool, Liverpool, UK   ² University College London, UK )
Purpose: Retention time (time to treatment failure) is a commonly used outcome in antiepileptic drug studies. In this paper we present the issues involved and demonstrate the importance of a full investigation of this outcome.
Method: Fifteen antiepileptic drug monotherapy trials and an add-on therapy clinic cohort are used to illustrate both design and analysis issues. Cumulative incidence analysis is described and applied to a dataset comparing lamotrigine and topiramate. The results are compared to the more common approach using standard survival analysis methods.
Results: A nonsignificant difference in overall retention between lamotrigine and topiramate (logrank test statistic = 2.58, 1 degree of freedom, p = 0.108) masked highly significant differences in opposite directions between the drugs with respect to withdrawals due to side effects (Gray's test statistic = 12.26, 1 degree of freedom, p = 0.0005) and poor seizure control (Gray's test statistic = 34.58, 1 degree of freedom, p < 0.0001).
Conclusion: Retention time can be measured reliably but care is needed to collect sufficient information on reasons for drug withdrawal to allow a competing risks analysis. Important differences between the profiles of antiepileptic drugs may be missed unless appropriate statistical methods are used to fully investigate retention time. Cumulative incidence rather than logrank analysis is likely to be a more powerful approach for comparison of standard and new antiepileptic drugs.     

18.

Gene inactivation of Na+/H+ exchanger isoform 1 attenuates apoptosis and mitochondrial damage following transient focal cerebral ischemia     

Wang Y  Luo J  Chen X  Chen H  Cramer SW  Sun D 《The European journal of neuroscience》2008,28(1):51-61

We investigated mechanisms underlying the Na⁺/H⁺ exchanger isoform 1 (NHE1)-mediated neuronal damage in transient focal ischemia. Physiological parameters, body and tympanic temperatures, and regional cerebral blood flow during 30 min of middle cerebral artery occlusion were similar in wild-type NHE1 (NHE1^+/+) and NHE1 heterozygous (NHE1^+/−) mice. NHE1^+/+ mice developed infarct volume of 57.3 ± 8.8 mm³ at 24 h reperfusion (Rp), which progressed to 86.1 ± 10.0 mm³ at 72 h Rp. This delayed cell death was preceded by release of mitochondrial cytochrome c (Cyt. C), nuclear translocation of apoptosis-inducing factor (AIF), activation of caspase-3, and TUNEL-positive staining and chromatin condensation in the ipsilateral hemispheres of NHE1^+/+ brains. In contrast, NHE1^+/− mice had a significantly smaller infarct volume and improved neurological function. A similar neuroprotection was obtained with NHE1 inhibitor HOE 642. The number of apoptotic cells, release of AIF and Cyt. C or levels of active caspase-3 was significantly reduced in NHE1^+/− brains. These data imply that NHE1 activity may contribute to ischemic apoptosis. Ischemic brains did not exhibit changes of NHE1 protein expression. In contrast, up-regulation of NHE1 expression was found in NHE1^+/+ neurons after in vitro ischemia. These data suggest that NHE1 activation following cerebral ischemia contributes to mitochondrial damage and ischemic apoptosis.     

19.

Inhibition by mitoxantrone of in vitro migration of immunocompetent cells: a possible mechanism for therapeutic efficacy in the treatment of multiple sclerosis     

Kopadze T  Dehmel T  Hartung HP  Stüve O  Kieseier BC 《Archives of neurology》2006,63(11):1572-1578

BACKGROUND: Damage of the blood-brain barrier and invasion of immunocompetent cells into the central nervous system represent key events in the immunopathogenesis of multiple sclerosis. Mitoxantrone hydrochloride reduces progression of disability and clinical exacerbations in patients with multiple sclerosis. Its precise mode of action is unclear. OBJECTIVE: To investigate the effects of mitoxantrone on the migratory capacity of immunocompetent cells ex vivo and in vitro. DESIGN: Case-control study. SETTING: Department of Neurology, Heinrich Heine University, Düsseldorf, Germany. PARTICIPANTS: Peripheral blood mononuclear cells (PBMCs) were obtained from 11 patients with multiple sclerosis before and after intravenous mitoxantrone treatment; PBMCs from 5 healthy control donors were treated with mitoxantrone in vitro. MAIN OUTCOME MEASURES: The migratory capacity was studied in an in vitro Boyden chamber assay; cells and their rates of migration were analyzed by light microscopy and flow cytometry. To determine the specificity of our findings, PBMCs were treated with perfosfamide in vitro. RESULTS: Mitoxantrone decreased the migratory capacity of CD14(+) monocytes and (to a lesser degree) of CD4(+) and CD8(+) T lymphocytes. These observations were confirmed when control PBMCs were treated with an equivalent dose of mitoxantrone in vitro. Similar effects were seen when PBMCs were preincubated with perfosfamide. The inhibitory effects of mitoxantrone on the migratory capacity of PBMCs were mediated by reduced matrix metalloproteinase 9 activity, as demonstrated by zymography, polymerase chain reaction, and inhibitory studies. CONCLUSION: Mitoxantrone may inhibit the migration of inflammatory cells into and within the central nervous system.     

20.

pSTAT1, pSTAT3, and T-bet as markers of disease activity in chronic inflammatory demyelinating polyradiculoneuropathy     

Francesca Madia  Giovanni Frisullo  Viviana Nociti  Amelia Conte  Marco Luigetti  Alessandra Del Grande  Agata Katia Patanella  Raffaele Iorio  Pietro Attilio Tonali  Anna Paola Batocchi  Mario Sabatelli 《Journal of the peripheral nervous system : JPNS》2009,14(2):107-117

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is considered an auto-immune disorder. We evaluated expression of pSTAT1, T-bet, and pSTAT3 in circulating T-cells, B-cells, and monocytes and spontaneous production of interleukin-17 (IL17), interferon-gamma (IFNγ), and interleukin-10 (IL10) by peripheral blood mononuclear cells (PBMCs) from 14 active CIDP patients compared with 6 patients with long-lasting remission and 20 controls. Active disease patients showed higher pSTAT1, T-bet, and pSTAT3 in CD4⁺ T-cells than controls (p < 0.001, p = 0.0002, p = 0.0097, respectively) and remission patients (p < 0.001, p = 0.0036, p = 0.0008, respectively). pSTAT1, T-bet, and pSTAT3 were also higher in monocytes from active CIDP patients than controls (p = 0.0011, p = 0.0041, p = 0.0413, respectively) and remission patients (p = 0.0073, p = 0.0274, p = 0.0251, respectively). Moreover in CD8⁺ T-cells, pSTAT3 expression was higher in active CIDP patients than in remission patients (p = 0.0345) and in controls (p = 0.0023). IL17 and IFNγ production were significantly higher in active CIDP patients than in controls (p < 0.0395, p = 0.0010, respectively); IFNγ levels were higher also in remission CIDP patients (p = 0.0073). IL10 levels were higher in active phase patients than in controls (p = 0.0334). Our data suggest that pSTAT1, T-bet, and pSTAT3 can be considered putative markers of disease activity and potential targets for specific therapies.     

IGF-I promotes neuronal migration and positioning in the olfactory bulb and the exit of neuroblasts from the subventricular zone

While insulin-like growth factor-I (IGF-I) supports neuronal and glial differentiation in the CNS, it is largely unknown whether IGF-I also influences neuronal migration and positioning. We show here that the pattern of olfactory bulb (OB) layering is altered in Igf-I ^−/− mice. In these animals, Tbr1⁺-glutamatergic neurons are misplaced in the mitral cell layer (ML) and the external plexiform layer (EPL). In addition, there are fewer interneurons in the glomerular layer and the EPL of the Igf-I ^−/− mice, and fewer newborn neurons are incorporated into the OB from the forebrain subventricular zone (SVZ). Indeed, neuroblasts accumulate in the postnatal/adult SVZ of Igf-I ^−/− mice. Significantly, the positioning of Tbr1⁺-cells in a primitive ML is stimulated by IGF-I in cultured embryonic OB slices, an effect that is partially repressed by the phosphoinositide 3-kinase (PI3K) inhibitor. In OB cell cultures, IGF-I increases the phosphorylation of disabled1 (P-Dab1), an adaptor protein that is a target of Src family kinases (SFK) in the reelin signalling pathway, whereas reduced P-Dab1 levels were found in Igf-I ^−/− mice. Neuroblast migration from the rostral migratory stream (RMS) explants of postnatal Igf-I ^−/− was similar to that from Igf-I ^+/+ explants. However, cell migration was significantly enhanced by IGF-I added to the explants, an effect that was repressed by PI3K and SFK inhibitors. These findings suggest that IGF-I promotes neuronal positioning in the OB and support a role for IGF-I in stimulating neuroblast exit from the SVZ into the RMS, thereby promoting the incorporation of newly formed neurons into the OB.     

T cell subsets in multiple sclerosis: a serial study

The relevance of abnormalities in the distribution of peripheral blood T lymphocyte subsets to the clinical manifestations of multiple sclerosis is not firmly established. A clinical and immunological follow-up of relapsing-remitting multiple sclerosis patients was performed in order to study the relationship of immune changes with the clinical course of the disease. Twenty patients were monitored monthly during a mean time of nine months for peripheral blood lymphocyte subsets (CD3, CD4, CD8, CD19), including the immunoregulatory subsets CD4CD29 (helper-inducer), and CD4CD45RA (suppressor-inducer) and activated T helper cells (CD4CD25) by flow cytometry. A total of 14 untreated relapses was included. The most significant observations were a decrease in T suppressor-inducer CD4⁺ CD45RA⁺ subset during clinical relapses ( P = 0.028) that was also detectable one month before ( P = 0.020) and the lack of changes in CD4⁺ CD29⁺ and CD8⁺ T cells. In addition, variations in the percentage of CD4⁺CD25⁺ activated T helper cells were not associated with clinical exacerbations. These results indicate the existence of a temporal association of immune changes in peripheral blood, but not activation, with the clinical manifestations of multiple sclerosis.     

Expression of chemokine receptors on peripheral blood mononuclear cells of patients with immune-mediated neuropathies treated with intravenous immunoglobulins

Intravenous immunoglobulin (IVIg) is an efficacious treatment for immune-mediated neuropathies like Guillain–Barré syndrome (GBS), chronic inflammatory demyelinating neuropathy (CIDP), and multifocal motor neuropathy (MMN). In the pathogenesis of immune-mediated neuropathies chemokines and their receptors play a crucial role. Using flow cytometry we examined whether IVIg modulates chemokine expression repertoires of T cells and monocytes. The expression of inflammatory chemokine receptors CCR1, CCR2, CCR4, CCR5, CCR6 and CXCR3 was investigated on circulating T-cell subsets, and CCR1, CCR2 and CCR5 on circulating monocytes before and after IVIg treatment in patients with immune-mediated neuropathies (MMN, n  = 7; GBS, n  = 1; CIDP, n  = 2). Furthermore, the homing potential of T cells was analyzed by the expression of CCR7, a chemokine receptor known to be utilized by mature T cells to recirculate into secondary lymphoid organs. In contrast to studies in chronic heart failure, no differences in expression patterns before and after IVIg treatment of any of the investigated chemokine receptors were found. Furthermore, the proportion of CD45RO-positive CD4⁺ or CD8⁺ T-cell subsets was not changed by IVIg treatment. Thus, we concluded that modulation of the expression of chemokine receptors on circulating leukocytes by IVIg is not a mode of action in immune-mediated neuropathies.     

Tuesday July 4, 200612:00–13:30Hall 1Platform SessionDrug Therapy II

¹ P. Williamson , ¹ C. Tudur Smith , ² J. Sander , and ¹ A. Marson (   ¹ University of Liverpool, Liverpool, UK   ² University College London, UK )
Purpose: Retention time (time to treatment failure) is a commonly used outcome in antiepileptic drug studies. In this paper we present the issues involved and demonstrate the importance of a full investigation of this outcome.
Method: Fifteen antiepileptic drug monotherapy trials and an add-on therapy clinic cohort are used to illustrate both design and analysis issues. Cumulative incidence analysis is described and applied to a dataset comparing lamotrigine and topiramate. The results are compared to the more common approach using standard survival analysis methods.
Results: A nonsignificant difference in overall retention between lamotrigine and topiramate (logrank test statistic = 2.58, 1 degree of freedom, p = 0.108) masked highly significant differences in opposite directions between the drugs with respect to withdrawals due to side effects (Gray's test statistic = 12.26, 1 degree of freedom, p = 0.0005) and poor seizure control (Gray's test statistic = 34.58, 1 degree of freedom, p < 0.0001).
Conclusion: Retention time can be measured reliably but care is needed to collect sufficient information on reasons for drug withdrawal to allow a competing risks analysis. Important differences between the profiles of antiepileptic drugs may be missed unless appropriate statistical methods are used to fully investigate retention time. Cumulative incidence rather than logrank analysis is likely to be a more powerful approach for comparison of standard and new antiepileptic drugs.     

Gene inactivation of Na+/H+ exchanger isoform 1 attenuates apoptosis and mitochondrial damage following transient focal cerebral ischemia

We investigated mechanisms underlying the Na⁺/H⁺ exchanger isoform 1 (NHE1)-mediated neuronal damage in transient focal ischemia. Physiological parameters, body and tympanic temperatures, and regional cerebral blood flow during 30 min of middle cerebral artery occlusion were similar in wild-type NHE1 (NHE1^+/+) and NHE1 heterozygous (NHE1^+/−) mice. NHE1^+/+ mice developed infarct volume of 57.3 ± 8.8 mm³ at 24 h reperfusion (Rp), which progressed to 86.1 ± 10.0 mm³ at 72 h Rp. This delayed cell death was preceded by release of mitochondrial cytochrome c (Cyt. C), nuclear translocation of apoptosis-inducing factor (AIF), activation of caspase-3, and TUNEL-positive staining and chromatin condensation in the ipsilateral hemispheres of NHE1^+/+ brains. In contrast, NHE1^+/− mice had a significantly smaller infarct volume and improved neurological function. A similar neuroprotection was obtained with NHE1 inhibitor HOE 642. The number of apoptotic cells, release of AIF and Cyt. C or levels of active caspase-3 was significantly reduced in NHE1^+/− brains. These data imply that NHE1 activity may contribute to ischemic apoptosis. Ischemic brains did not exhibit changes of NHE1 protein expression. In contrast, up-regulation of NHE1 expression was found in NHE1^+/+ neurons after in vitro ischemia. These data suggest that NHE1 activation following cerebral ischemia contributes to mitochondrial damage and ischemic apoptosis.     

Inhibition by mitoxantrone of in vitro migration of immunocompetent cells: a possible mechanism for therapeutic efficacy in the treatment of multiple sclerosis

BACKGROUND: Damage of the blood-brain barrier and invasion of immunocompetent cells into the central nervous system represent key events in the immunopathogenesis of multiple sclerosis. Mitoxantrone hydrochloride reduces progression of disability and clinical exacerbations in patients with multiple sclerosis. Its precise mode of action is unclear. OBJECTIVE: To investigate the effects of mitoxantrone on the migratory capacity of immunocompetent cells ex vivo and in vitro. DESIGN: Case-control study. SETTING: Department of Neurology, Heinrich Heine University, Düsseldorf, Germany. PARTICIPANTS: Peripheral blood mononuclear cells (PBMCs) were obtained from 11 patients with multiple sclerosis before and after intravenous mitoxantrone treatment; PBMCs from 5 healthy control donors were treated with mitoxantrone in vitro. MAIN OUTCOME MEASURES: The migratory capacity was studied in an in vitro Boyden chamber assay; cells and their rates of migration were analyzed by light microscopy and flow cytometry. To determine the specificity of our findings, PBMCs were treated with perfosfamide in vitro. RESULTS: Mitoxantrone decreased the migratory capacity of CD14(+) monocytes and (to a lesser degree) of CD4(+) and CD8(+) T lymphocytes. These observations were confirmed when control PBMCs were treated with an equivalent dose of mitoxantrone in vitro. Similar effects were seen when PBMCs were preincubated with perfosfamide. The inhibitory effects of mitoxantrone on the migratory capacity of PBMCs were mediated by reduced matrix metalloproteinase 9 activity, as demonstrated by zymography, polymerase chain reaction, and inhibitory studies. CONCLUSION: Mitoxantrone may inhibit the migration of inflammatory cells into and within the central nervous system.     

pSTAT1, pSTAT3, and T-bet as markers of disease activity in chronic inflammatory demyelinating polyradiculoneuropathy

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is considered an auto-immune disorder. We evaluated expression of pSTAT1, T-bet, and pSTAT3 in circulating T-cells, B-cells, and monocytes and spontaneous production of interleukin-17 (IL17), interferon-gamma (IFNγ), and interleukin-10 (IL10) by peripheral blood mononuclear cells (PBMCs) from 14 active CIDP patients compared with 6 patients with long-lasting remission and 20 controls. Active disease patients showed higher pSTAT1, T-bet, and pSTAT3 in CD4⁺ T-cells than controls (p < 0.001, p = 0.0002, p = 0.0097, respectively) and remission patients (p < 0.001, p = 0.0036, p = 0.0008, respectively). pSTAT1, T-bet, and pSTAT3 were also higher in monocytes from active CIDP patients than controls (p = 0.0011, p = 0.0041, p = 0.0413, respectively) and remission patients (p = 0.0073, p = 0.0274, p = 0.0251, respectively). Moreover in CD8⁺ T-cells, pSTAT3 expression was higher in active CIDP patients than in remission patients (p = 0.0345) and in controls (p = 0.0023). IL17 and IFNγ production were significantly higher in active CIDP patients than in controls (p < 0.0395, p = 0.0010, respectively); IFNγ levels were higher also in remission CIDP patients (p = 0.0073). IL10 levels were higher in active phase patients than in controls (p = 0.0334). Our data suggest that pSTAT1, T-bet, and pSTAT3 can be considered putative markers of disease activity and potential targets for specific therapies.