Tony Sehr

Letter to the editor on the paper: “The majority of natalizumab-treated MS patients have high natalizumab concentrations at time of re-dosing”

We therefore propose to clarify three issues in future studies. First, as Yates et al.3 did not perform immunostaining of CD4+ T cells, the presence or absence of CD4+ T cells around small cortical veins should be elucidated. Second, the exact location of fibrin(ogen) deposition in the MS cortex remains unclear. It is critical to clarify whether fibrin(ogen) is deposited around small cortical veins. Third, an association of any HLA class I gene allele with cortical lesions has never been reported, and therefore, we believe it is worth studying in the future.

Dendritic cells are specifically affected by different immunomodulatory treatment strategies in multiple sclerosis

Objectives: The incidence of bacterial infections of the central nervous system (CNS) in adults increases with age, and the clinical outcome in elderly individuals is worse than in young persons. Death in the acute phase of the infection and neurologic or neuropsychologic deficits are common complications, and better therapies are needed. Since microglial cells play a key role for the defence of the brain against bacterial infections, we investigated age-related functional changes of these cells in vitro. Methods: Primary microglial cultures were obtained from brains of C57BL/6-N mice aged 2 months (young) and 18 months (aged). For activation, cells cultured in 96-well-plates were treated with agonists of Toll-like receptor (TLR) 1/2 [Tripalmitoyl-S-glycerylcysteine (Pam3CSK4); 0.1 μg/ml] and TLR 4 [endotoxin (LPS) from Escherichia coli serotype 026:B6; 0.01 μg/ml] for 24 h in the presence of interferon-gamma (IFN-gamma; 100 U/ml). Non-activated control cells were treated with IFN-gamma only. Young and aged microglial cells were compared regarding their release of nitric oxide (NO), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and CXCL1 (KC) into the cell culture supernatant. Results: Upon stimulation with agonists of TLR 2 and 4, aged microglial cells released significantly less NO and cytokines/ chemokines than young microglial cells: nitrite upon Pam3CSK4 treatment: 6.97 (5.96/8.28) vs. 18.40 (8.78/20.89) μM, p = 0.008; TNF-alpha upon LPS treatment: 284.10 (262.40/300.50) vs. 681.20 (468.50/1024.00) pg/ml, p = 0.002; IL-6 upon Pam3CSK4 treatment: 85.70 (17.86/144.30) vs. 924.90 (82.00/937.90) pg/ml, p = 0.04; KC upon LPS treatment: 15.63 (15.63/24.23) vs. 75.00 (44.10/117.40) pg/ml, p = 0.007 [representative results, concentrations in the cell culture supernatant indicated as median (25./75. percentile), comparison aged vs. young by Mann–Whitney-U-test, n = 5–8]. Conclusion: Our in-vitro data show a reduced release of NO and inflammatory cytokines/chemokines by aged microglial cells upon activation with bacterial TLR agonists. The age-related decline of microglial functions might contribute to the higher susceptibility of elderly individuals to bacterial CNS infections. Strategies to improve the functions of aged microglial cells appear promising for prevention and treatment of CNS infections in elderly patients.

Monitoring Free and Cell-Bound Natalizumab in CSF and Blood in Natalizumab-Treated MS Patients (P02.071)

Objective: To develop a FACS-based assay to detect free and cell-bound natalizumab-concentration in CSF and blood. Background Natalizumab is an approved therapy for highly active RRMS. By blocking VLA-4 on leukocytes, migration of immune cells into the CNS is reduced. Whereas the blockade of VLA-4 in the periphery is well known, no data are available on pharmacodynamics and -kinetics of natalizumab in the CSF. Design/Methods: For the analysis of natalizumab concentration/surface binding and VCAM-binding in CSF and blood, paired CSF and blood samples of natalizumab-treated MS patients were prospectively analyzed. Results: In patients treated with natalizumab, significant amounts of natalizumab (0.047± 0.040µg/ml) could be found in CSF, about 450-times lower than in serum (21.78± 14.4µg/ml). In controls no natalizumab was detectable in CSF nor serum. Concentration in CSF depends on BBB-damage and individual properties, as demonstrated by correlation analysis. Functional experiments showed, that natalizumab in CSF and serum was still functional active on VLA-4-blocking. Quantity of cell-bound natalizumab differed between individual cell-subsets. Before natalizumab-infusion mean saturation in relation to maximal load differed between 48% to 62% before and 74% to 92% after infusion. We could find a strong correlation between cell-bound and free natalizumab. Relative decrease 4 weeks after infusion was more pronounced for free (-78%) than cell-bound natalizumab (-25%). In-vitro experiments demonstrated that cell-bound natalizumab could serve as a reservoir. Patients with anti-natalizumab neutralizing antibodies could be easily detected by analysis of natalizumab-concentration in serum and CSF. Here no natalizumab could be demonstrated. In-vitro, several neutralizing antibodies were able to remove cell-bound natalizumab from immune cells. Conclusions: Here we demonstrate for the first time that still functionally active natalizumab was detectable in the CSF of natalizumab-treated patients. Using our assay, we were able to describe individual pharmacokinetics and -dynamics in natalizumab-treated patients. Our approach could be used to monitor prospectively natalizumab-treated patients. Disclosure: Dr. Sehr has nothing to disclose. Dr. Hainke has nothing to disclose. Dr. Thomas has received research support from Teva Neuroscience. Dr. Schultheiss has nothing to disclose. Dr. Ziemssen has received personal compensation for activities with Biogen Idec, Bayer Healthcare, Novartis, Sanofi-Aventis Pharmaceuticals, Inc., Teva Neuroscience, and Synthon. Dr. Ziemssen has received research support for from Bayer Healthcare, Biogen Idec, Novartis, Teva Neuroscience, and Sanofi-Aventis Pharmaceuticals, Inc.

Pharmacokinetics and -Dynamics of Natalizumab after Cessation and at Different Dosing Intervals (P06.167)

Objective: To assess the pharmacokinetics and -dynamics after cessation of natalizumab and at different application intervals. Background Natalizumab, a humanized recombinant monoclonal antibody, is approved for the treatment of highly active relapsing-remitting multiple sclerosis. As decreased immune surveillance of the brain can cause progressive multifocal leukoencephalopathy (PML) during natalizumab treatment, treatment concepts of treatment holidays and different treatment intervals beyond 4 weeks have been proposed. Up to now, there are no data available on immunology and natalizumab concentration in blood and CSF. Design/Methods: Patients were analyzed during cessation of natalizumab and at different infusion intervals of natalizumab (every 4, 5 or 8 weeks) using FACS analysis of blood and CSF at different timepoints. In addition, free and cell-bound quantities of natalizumab were measured. Results: After cessation of natalizumab plasma concentrations decreased by 54% in month 1, 90% in month 2 and 99,7% in month 3 and 4. We could observe cell-bound natalizumab in blood 2 up to 4 months after stop of treatment. Blood and CSF analysis in patients with relapses after cessation of natalizumab demonstrated no cell-bound natalizumab and plasma concentrations which were decreased to 99% from baseline levels. We could demonstrate different pre infusion plasma concentrations of natalizumab for treatment intervals of 4 (25±5 µg/ml), 5 (15±5 µg/ml) or 8 weeks (6±5µg/ml). Post infusion natalizumab levels ranged from 60µg/ml up to 130µg/ml independent of the respective treatment interval. Regarding pre-infusion cell-bound natalizumab, we could describe MFI of 3500-6000 for 4 weeks interval, 1000-2500 for 5 and 1000-1200 for 8 weeks treatment interval. Conclusions: Monitoring pharmakokinetics and –dynamics can be helpful in analysis of treatment holidays and different application intervals of natalizumab. Supported by: Biogen Idec. Disclosure: Dr. Hainke has nothing to disclose. Dr. Ziemssen has received personal compensation for activities with Biogen Idec, Bayer Healthcare, Novartis, Sanofi-Aventis Pharmaceuticals, Inc., Teva Neuroscience, and Synthon. Dr. Ziemssen has received research support for from Bayer Healthcare, Biogen Idec, Novartis, Teva Neuroscience, and Sanofi-Aventis Pharmaceuticals, Inc. Dr. Sehr has nothing to disclose. Dr. Thomas has received research support from Teva Neuroscience. Dr. Schultheiss has nothing to disclose.