Neuroscience Letters 406 (2006) 270–275
`
`Effect of glatiramer acetate (Copaxone®) on the immunophenotypic and
`cytokine profile and BDNF production in multiple sclerosis:
`A longitudinal study
`Y. Blanco a,b, E.A. Moral c,d, M. Costa e, M. G´omez-Choco a,b, J.F. Torres-Peraza b,f,
`L. Alonso-Magdalena c,d, J. Alberch b,f, D. Jaraquemada e, T. Arbizu c,d,
`F. Graus a,b, A. Saiz a,b,∗
`
`a Service of Neurology, Hospital Cl´ınic, Universitat de Barcelona, Villarroel 170, 08036 Barcelona, Spain
`b Institut d’Investigaci´o Biom`edica August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Villarroel 170, 08036 Barcelona, Spain
`c Service of Neurology, Hospital Universitario de Bellvitge, Avda. Feixa Llarga s/n, 08907, L’Hospitalet de Llobregat, Barcelona, Spain
`d Institut dˇıInvestigaci´o Biom`edica Bellvitge (IDIBELL), Avda. Feixa Llarga s/n, 08907, L’Hospitalet de Llobregat, Barcelona, Spain
`e Immunology Unit, Institut de Biotecnologia i Biomedicina, Universitat Aut`onoma de Barcelona, Spain
`f Department of Cell Biology and Pathology, Universitat de Barcelona, Barcelona, Spain
`Received 2 June 2006; received in revised form 11 July 2006; accepted 21 July 2006
`
`Abstract
`
`We assessed the effect of glatiramer acetate (GA) on the immunophenotypic and cytokine profile and the BDNF production by peripheral
`blood mononuclear cells, and their association with the clinical response in 19 na¨ıve-treated MS patients prospectively followed-up after GA
`therapy. Two patients withdrew the therapy. After a median follow-up of 21 months, twelve were considered responders and five as non-responders.
`Non-responder patients had significant longer disease duration and a higher EDSS score at baseline. In the responder group, a significant decrease
`in the percentage of INF-␥ producing total lymphocytes, CD4+ and CD8+ T cells, and reduced percentage of IL-2 producing CD4+ and CD8+ T
`cells were observed at 12, 18 and 24 months. These changes were associated with a significant increase in the percentage of CD3+, CD4+ and
`CD4+CD45RA+ T cells, and BDNF production from month 6 that remained significant throughout the study. We did not observe significant changes
`in the nonresponder group for any of the parameters studied. Our data suggest that GA treatment induces a downmodulation of proinflammatory
`cytokines associated with the regulation of the peripheral T cell compartment and with increased production of BDNF that might be related to the
`clinical response.
`© 2006 Elsevier Ireland Ltd. All rights reserved.
`
`Keywords: Multiple sclerosis; Glatiramer acetate; BDNF; Cytokines; Lymphocyte immunophenotyping; Immunomodulatory treatment
`
`Glatiramer acetate (GA, Copaxone®) is an approved agent for
`the immunomodulatory treatment of relapsing-remitting mul-
`tiple sclerosis (RRMS). Although the mechanisms of action
`of GA are not fully understood, recent studies support both
`immunomodulatory and neuroprotective effects of the drug
`[17,24]. Besides the induction of anergy in autoreactive T
`cells, GA induces a shift of GA-reactive T cells from a Th1
`to a Th2 phenotype [3,4,10,13,18] that are thought to release
`anti-inflammatory cytokines but also neurotrophic factors such
`
`∗
`
`Corresponding author at: Service of Neurology, Hospital Clinic, Villarroel
`170, Barcelona 08036, Spain. Tel.: +34 932275414; fax: +34 932275783.
`E-mail address: asaiz@clinic.ub.es (A. Saiz).
`
`0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
`doi:10.1016/j.neulet.2006.07.043
`
`as brain-derived neurotrophic factor (BDNF) within the CNS
`[1,3,25]. However, most of these immunological effects are
`based on the results of the analysis of GA-specific T-cell lines
`and less is known on the ex vivo systemic effects on periph-
`eral blood mononuclear cells (PBMC) [9,11,15,16,23]. The aim
`of the present study was to assess the effect of GA on the
`immunophenotypic and cytokine profile and the BDNF produc-
`tion by PBMC, and to analyze the association of these param-
`eters with the clinical response in a group of RRMS patients
`prospectively followed-up after GA treatment.
`Nineteen na¨ıve-treated patients with RRMS according to the
`Poser criteria [19] from the MS Units of the two participant
`hospitals were included in the study. All patients began treat-
`ment with GA because they had active disease with two or
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`
`Table 1
`Baseline clinical characteristics of responder and non-responder patients to GA therapy
`
`Overall
`
`Responders
`
`Non-respondersa
`
`pb
`
`19
`14/5
`34.7± 9.4
`5.6± 3.3
`1.2± 0.6
`1.5± 1.3
`
`12
`9/3
`34.8± 8.0
`3.8± 0.9
`1.2± 0.6
`1.4± 0.8
`
`more relapses in the previous two years and with an Expanded
`Disability Status Scale (EDSS) score between 0 and 5.5. Base-
`line clinical characteristics are summarized in Table 1. Patients
`were regularly followed every 3 months and additional assess-
`ments were done in the event of a relapse. During the follow-up,
`patients who presented identical or higher annual relapse rate
`and/or increase of at least 1 EDSS point confirmed at six months,
`were defined as nonresponders to GA. PBMC were collected
`before treatment, at 6 months after GA therapy and then every 6
`months. In case of coincidental relapse, the sample was taken 1
`month after steroid administration. The study was approved by
`the Ethical Committee of the two participating hospitals.
`PBMC phenotypical characterization was analyzed using a
`FACSCalibur flow cytometer (Becton Dickinson Immunocy-
`tometry Sistems (BDIS), San Jos´e, CA, USA), as previously
`reported [2]. Monoclonal antibodies to the following surface
`markers were used: CD3, CD8, CD4, CD19, HLA-DR, CD62-
`L, CD45RA, CD45RO, (BD Biosciences, San Jose, CA, USA),
`and CD25 (BD, Pharmingen, San Diego, CA, USA).
`Intracellular cytokine production was detected by flow
`cytometry as previously described [2,7] with slight modifi-
`cations. Briefly, fresh peripheral blood was incubated for 4 h
`with phorbol 12-myristate (PMA) (25 ng/ml, Sigma–Aldrich,
`St. Louis) and ionomycin (1 ␮g/ml, Sigma–Aldrich) to stim-
`ulate cytokine production. Stimulated and unstimulated cells
`were treated with Brefeldin A (10 ␮g/ml, Sigma–Aldrich), an
`inhibitor of secretion, followed by superficial staining, cell fix-
`ation, permeabilization, and then intracytoplasmic staining for
`detection of the accumulated cytokines. T cells were first gated
`by their expression of CD8 or CD4 followed by the detection of
`IFN-␥, IL-2 (as markers of Th1 phenotype) and IL-4 (as marker
`of Th2 phenotype) (BD, Pharmingen, San Diego, CA, USA)
`positive cells.
`BDNF release levels were measured in the supernatants from
`PBMC unstimulated and stimulated with anti-CD3 and soluble
`anti-CD28 antibodies by using an ELISA kit (Promega, Madi-
`son, USA) as previously described [2].
`Non-parametric tests were used for all data comparisons:
`Friedman and Wilcoxon signed ranks tests for paired patients
`groups. Pearson’s correlation coefficient was used for the corre-
`lation analysis. Significance levels were set at 5% (p < 0.05).
`Two patients withdrew the treatment because of adverse
`events in the first 3 months. After a median follow-up of 21
`months, twelve patients were considered as responders and 5 as
`nonresponders. Responder patients had a decrease of the annu-
`
`5
`4/1
`40.2± 11.3
`8.1± 2.6
`1.0± 0.3
`3.1± 1.2
`
`ns
`ns
`<0.0001
`ns
`<0.0001
`
`Number of patients
`Gender (W/M)
`Age (mean± S.D.; years)
`Disease duration (mean± S.D.; years)
`Annual relapse rate (mean± S.D.; in prior 2 years)
`Baseline EDSS (mean± S.D.)
`a Two patients withdrew the treatment in the first 3 months.
`b Refers to comparison of clinical variables between responder and non-responder patients.
`alized relapse rate (0.3± 0.4 during treatment versus 1.2± 0.6
`before treatment) and did not present significant changes in the
`EDSS score (1.4± 0.8 at initiation of treatment versus 1.6± 0.8
`at last follow-up). Non-responders had a mean of 1.3± 0.9 annu-
`alized relapse rate (1.0± 0.3 before treatment), and a mean
`increase of 0.6± 0.9 in the EDSS score (3.1± 1.2 at initiation
`of treatment versus 4.1± 1.4 at last follow-up). The only sig-
`nificant difference between both groups was that nonresponders
`patients had longer disease duration and a higher EDSS score at
`initiation of GA therapy (p < 0.0001) (Table 1).
`Results of the immunophenotypic and cytokine profile along
`the evolution are presented as mean± standard deviation in
`Tables 2 and 3 for both groups. There were no significant
`baseline differences between both groups of patients for any
`of the parameters studied. After GA therapy a significant
`increase in the percentage of CD3+, CD4+ and CD4+CD45RA+
`(na¨ıve) T cells was seen at 6, 12, 18 and 24 months in
`the responder group of patients compared with that found
`before treatment (p =0.003, p = 0.012, p = 0.05; p = 0.002,
`p = 0.01, p = 0.038; p = 0.002, p = 0.005, p = 0.036; p = 0.021,
`p = 0.05, p = 0.028; respectively). The na¨ıve/memory CD4+ T
`cells (CD4+CD45RA+/CD4+CD45RO+) ratio also increased
`at 12 months and remained significant during the follow-up
`(p = 0.028, p = 0.026, p = 0.011). No significant changes were
`observed in the percentage of CD4+CD25+ T cells. No sig-
`nificant modification in T cells population frequencies were
`detected over the study period in the nonresponder group as
`compared with baseline values.
`A significant decrease in the number of IFN-␥ producing
`CD3+, CD4+ and CD8+ T cells was observed in the respon-
`der group at 12, 18 and 24 months after GA therapy, com-
`pared with pretreatment values (p =0.028, p = 0.008, p = 0.008;
`p = 0.024, p = 0.008, p = 0.011; p = 0.012, p = 0.012, p = 0.012;
`respectively) (see Fig. 1 and Table 1). In the same way, the num-
`ber of IL-2 producing CD4+ and CD8+ T cells also decreased
`(p =0.013, p = 0.021; p = 0.037, p = 0.037; p = 0.036, p = 0.028;
`respectively). No significant changes were observed in the num-
`ber of IL-4 producing CD4+ and CD8+ T cells. In the nonre-
`sponder patients’ group, no significant differences were found
`over the study period as compared with baseline values.
`A significant increase of mean levels of BDNF in the super-
`natants of unstimulated PBMC was observed in the responder
`group from month 6 and throughout the follow-up after GA
`therapy as compared with baseline (Fig. 2). Similar results
`were found after stimulation with anti-CD3 and soluble anti-
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`Fig. 1. An example of the flow cytometry analysis of cytoplasmic IFN-␥ and IL-4 expression by CD4+ and CD8+ T cells of a responder patient before and after GA treatment, as described in methods. Quadrants
`were set based on the isotype control and on unstimulated samples. Values represent the percentage of positive cells.
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`Table 2
`Peripheral T cell population before and after GA treatment
`
`Y. Blanco et al. / Neuroscience Letters 406 (2006) 270–275
`
`273
`
`R
`NR
`
`R
`NR
`
`R
`NR
`
`R
`NR
`
`R
`NR
`
`R
`NR
`
`R
`NR
`
`R
`NR
`
`CD3+
`
`CD4+
`
`CD4+CD45RA+
`
`CD4+CD45RO+
`
`RA+/RO+ ratioa
`
`CD8+
`
`CD8+CD45RA+
`
`CD8+CD45RO+
`
`24 months
`18 months
`12 months
`6 months
`Baseline
`76.1 ± 3.3a
`71.0 ± 5.1
`75.7 ± 5.5a
`76.5 ± 4.8a
`75.2 ± 3.9a
`77.2 ± 1.2
`76.1 ± 1.1
`79.8 ± 3.6
`75.4 ± 3.3
`76.1 ± 7.4
`54.4 ± 7.8a
`53.7 ± 7.5a
`53.9 ± 7.7a
`53.3 ± 6.1a
`47.8 ± 9.1
`54.1 ± 9.9
`45.4 ± 11.9
`51.2 ± 8.9
`47.7 ± 5.2
`51.5 ± 6.1
`43.9 ± 10.1a
`35.3 ± 16.0
`42.9 ± 9.4a
`43.5 ± 10.2a
`44.8 ± 12.8a
`40.2 ± 18.4
`30.6 ± 21.5
`48.1 ± 17.5
`34.1 ± 12.2
`32.6 ± 15.0
`37.8 ± 9.0
`40.6 ± 9.1
`38.8 ± 8.1
`38.8 ± 6.5
`39.3 ± 8.0
`46.0 ± 9.4
`45.8 ± 11.0
`41.5 ± 17.8
`43.4 ± 7.4
`49.7 ± 14.0
`1.2 ± 0.4a
`1.1 ± 0.4a
`1.2 ± 0.4a
`1.2 ± 0.4
`0.9 ± 0.5
`0.9 ± 0.3
`0.7 ± 0.6
`1.1 ± 0.8
`0.7 ± 0.3
`0.8 ± 0.7
`21.3 ± 5.0
`21.2 ± 6.5
`21.0 ± 5.9
`22.5 ± 5.6
`21.0 ± 7.0
`24.1 ± 9.7
`30.7 ± 13.1
`27.4 ± 9.9
`26.2 ± 11.6
`26.7 ± 8.0
`57.8 ± 10.5
`57.2 ± 12.4
`60.2 ± 9.7
`6.3.3 ± 11.0
`58.4 ± 11.7
`59.6 ± 17.3
`49.0 ± 22.4
`54.5 ± 23.7
`58.1 ± 18.8
`53.7 ± 17.0
`23.5 ± 5.7
`24.8 ± 9.8
`21.7 ± 6.6
`24.2 ± 10.5
`23.9 ± 8.3
`23.0 ± 2.7
`19.8 ± 3.0
`17.8 ± 4.5
`22.9 ± 4.9
`23.2 ± 5.9
`31.3 ± 6.7
`28.5 ± 6.3
`28.7 ± 8.9
`31.5 ± 8.9
`31.7 ± 11.3
`R
`31.0 ± 5.1
`24.3 ± 6.4
`29.5 ± 4.8
`31.5 ± 8.5
`39.1 ± 6.3
`CD4+CD25+
`NR
`Results are expressed as mean± standard deviation of the percentage of positive cells for each surface marker; R = responder group; NR = non-responder group.
`a CD4+CD45RA+ to CD4+CD45RO+ T cells ratio.
`
`CD28 antibodies (data not shown). No significant changes were
`observed for the nonresponder group (Fig. 2).
`No significant correlation was found between basal immuno-
`logical data and clinical features (sex, age, disease duration,
`annualized relapse rate and EDSS score), nor there was any
`significant correlation between immunological measurements
`on treatment and clinical outcome (number of relapses, change
`of the annualized relapse rate, increase of the EDSS score
`and last EDSS score). None of the clinical and immunological
`data analyzed could significantly predict responsiveness to GA
`therapy.
`
`Our study shows that in vivo therapy with GA induces a down-
`modulation of proinflammatory cytokine-producing T cells in
`the periphery, and increases the BDNF production by unstimu-
`lated PBMC but also after stimulation involving T-cell receptor-
`mediated activation, and this effect is sustained over time but
`only observed in patients with clinical response to the drug.
`These data are in line with the results previously shown by
`the more complex method of analysis of GA-specific T-cell
`lines [1,3,4,10,13,18,25], although few immunological longi-
`tudinal studies with clinical correlation such as we present have
`been reported so far [22]. In this sense, other authors observed
`
`Table 3
`Intracellular cytokine staining profile before and after GA treatment
`
`12 m
`
`18 m
`
`IFN-␥ CD3+
`
`IFN-␥ CD4+
`
`IFN-␥ CD8+
`
`IL-2 CD4+
`
`IL-2 CD8+
`
`IL-4 CD4+
`
`R
`NR
`
`R
`NR
`
`R
`NR
`
`R
`NR
`
`R
`NR
`
`R
`NR
`
`6 m
`Baseline
`14.6 ± 5.5
`19.7 ± 9.8
`0.024
`0.028
`28.4 ± 11.4
`15.1 ± 12.6
`20.2 ± 17.1
`24.0 ± 17.6
`14.1 ± 8.7
`20.6 ± 9.4
`0.008
`0.008
`18.0 ± 5.8
`11.3 ± 2.5
`14.2 ± 8.9
`23.5 ± 12.4
`39.1 ± 14.8
`47.3 ± 18.1
`0.011
`0.008
`67.9 ± 14.8
`45.2 ± 16.3
`42.4 ± 30.2
`51.1 ± 27.5
`50.4 ± 15.4
`40.9 ± 17.5
`0.037
`0.013
`45.1 ± 12.4
`43.0 ± 14.6
`42.3 ± 5.6
`44.3 ± 11.9
`22.3 ± 9.6
`17.2 ± 11.5
`0.037
`0.021
`17.3 ± 3.2
`15.4 ± 2.8
`19.8 ± 3.5
`14.6 ± 2.6
`2.7 ± 1.1
`4.7 ± 2.5
`3.3 ± 0.8
`3.0 ± 2.0
`4.2 ± 2.5
`3.5 ± 1.1
`3.7 ± 3.4
`3.3 ± 1.3
`2.5 ± 1.5
`3.4 ± 2.7
`2.4 ± 1.4
`3.4 ± 1.2
`R
`2.9 ± 0.4
`1.8 ± 0.6
`2.5 ± 0.6
`2.0 ± 1.8
`IL-4 CD8+
`NR
`Results are expressed as mean± standard deviation of the percentage of cells staining positively. R = responder group; NR = non-responder group.
`
`24 m
`
`0.012
`20.9 ± 15.2
`0.012
`12.7 ± 9.7
`0.012
`49.9 ± 15.0
`0.036
`47.9 ± 11.7
`0.026
`13.9 ± 2.7
`1.1 ± 1.0
`3.8 ± 1.3
`1.7 ± 1.0
`1.0 ± 0.5
`
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`Fig. 2. BDNF levels in the supernatant of unstimulated PBMC of responder (left graph) and non-responder (right graph) group before and after GA treatment.
`Results are expressed in pg/ml. Time is expressed in months; time 0 refers to baseline. p-values are calculated in comparison with baseline and corrected for multiple
`comparisons. S.D.: standard deviation.
`
`an increase in the IFN-␥ secretion in responders to treatment;
`however the study was cross-sectional and retrospective [12].
`Interesting, our significant immune changes started at month 6
`for the immunophenotypic change and BDNF production, and
`at month 12 for the Th1 cytokine reduction, consistent with a
`delayed mechanism of action and a radiologic response, as was
`seen in the serial magnetic resonance imaging-based clinical
`trial [6].
`However, the most interesting data we found was that the
`reduction of Th1-type cell number was accompanied by an in
`vivo change in the peripheral T cell compartment characterized
`by a significant increase of CD4+CD45RA+ (na¨ıve) T cell fre-
`quency associated with an increased RA+/RO+ (na¨ıve/memory)
`ratio. A finding that extends a previous in vitro study that showed
`that the response to GA was driven by the CD4+CD45RA+ T
`cell subpopulation [23].
`The relevance of this increased frequency of peripheral
`CD4+CD45RA+ T cells after GA therapy to the clinical response
`we observed is intriguing. Nevertheless, in MS patients the na¨ıve
`T cell frequency is lower than average [8], and have a reduced
`TREC (TCR excision circles) content in circulating T cells,
`especially for CD4+ T cells, [14], suggesting that the release of
`na¨ıve T cells from the thymus could be impaired in MS. Because
`CD4+CD45RA+ T cells number as well as TCR levels decline
`with age [14,21] it could be that the results we found were related
`to the different, although non significant, age of both groups of
`patients. However, it is unlikely because we did not observe an
`increase in the frequency of CD4+CD45RA+ T cells in 4 addi-
`tional RRMS patients followed at least for 1 year after treatment
`with IFN-␤ who had a similar age (mean, 34.25± 7.93 years)
`to the GA responder group. Interesting, all four patients were
`considered as responders to IFN-␤ by using the same clinical
`definition.
`Finally, if the differences we found on GA therapy merely
`reflect that the two patient groups were in different stages of the
`disease is unknown. However, it is likely because similar clin-
`
`ical results have been observed with other immunomodulatory
`therapies that showed higher therapeutic benefit in patients with
`shorter disease duration and lesser disability [20,5].
`In conclusion, our study suggests that in addition to down-
`modulation of proinflammatory cytokines and increased secre-
`tion of BDNF, GA therapy may induce the regulation of the
`peripheral T cell compartment. Whether this regulation con-
`tributes to the clinical efficacy of GA needs further confirmatory
`studies in a larger patient sample.
`
`Acknowledgements
`
`Supported in part by Red CIEN, and grant from Instituto
`Carlos III (PI040659). M.G.-C. is a recipient of a postresidency
`grant from the Hospital Clinic, and J.F.T.-P. is a fellow from
`Fundaci´o La Caixa.
`
`References
`
`[1] R. Aharoni, B. Kayhan, R. Eilam, M. Sela, R. Arnon, Glatiramer acetate-
`specific T cells in the brain express T helper 2/3 cytokines and brain-
`derived neurotrophic factor in situ, Proc. Natl. Acad. Sci. U.S.A. 100 (2003)
`14157–14162.
`[2] Y. Blanco, A. Saiz, M. Costa, J.F. Torres-Peraza, E. Carreras, J. Alberch,
`D. Jaraquemada, F. Graus, Evolution of brain-derived neurotrophic factor
`levels after autologous hematopoietic stem cell transplantation in multiple
`sclerosis, Neurosci. Lett. 380 (2005) 122–126.
`[3] M. Chen, K. Conway, K.P. Johnson, R. Martin, S. Dhib-Jalbut, Sustained
`immunological effects of galtiramer acetate in patients with multiple scle-
`rosis treated for over 6 years, J. Neurol. Sci. 201 (2002) 71–77.
`[4] M. Chen, R.M. Valenzuela, S. Dhib-Jalbut, Glatiramer acetate-reactive T
`cells produced brain-derived neurotrophic factor, J. Neurol. Sci 215 (2003)
`37–44.
`[5] A.J. Coles, J. Deans, A. Compston, Campath-1 H treatment of multiple
`sclerosis: lessons from the bedside for the bench, Clin. Neurol. Neurosurg.
`106 (2004) 270–274.
`[6] G. Comi, M. Filippi, J.S. Wolinsky, The European/Canadian Glatiramer
`Acetate Study Group, European/Canadian multicenter, double-blind, ran-
`domized, placebo-controlled study of the effects of glatiramer acetate
`
`Page 5 of 6
`
`YEDA EXHIBIT NO. 2125
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`Y. Blanco et al. / Neuroscience Letters 406 (2006) 270–275
`
`275
`
`on magnetic resonance imaging-measured disease activity and burden in
`patients with relapsing multiple sclerosis, Ann. Neurol. 49 (2001) 290–297.
`[7] M. Costa, A. Saiz, R. Casmitjana, M.F. Casta˜ner, A. Sanmart´ı, F. Graus, D.
`Jaraquemada, T-cell reactivity to glutamic acid decarboxylase in stiff-man
`s´ındrome and cerebellar ataxia associated with polyendocrine autoimmu-
`nity, Clin. Exp. Immunol. 192 (2002) 471–478.
`[8] B. Crucian, P. Dunne, H. Friedman, R. Ragsdale, S. Pross, R. Widen,
`−
`/CD28+ suppressor cell precursor and
`Alterations in levels of CD28
`CD45RO+/CD4+ memory T lymphocytes in the peripheral blood of mul-
`tiple sclerosis patients, Clin. Diagn. Lab. Immunol. 2 (1995) 249–252.
`[9] S. Dhib-Jalbut, M. Chen, A. Said, M. Zhan, K.P. Johnson, R. Martin,
`Glatiramer acetate-reactive peripheral blood mononuclear cells respond
`to multiple sclerosis myelin antigens with a Th2-biased phenotype, J. Neu-
`roimmunol. 140 (2003) 163–171.
`[10] P.W. Duda, M.C. Schmied, S.L. Cook, J.I. Krieger, D.A. Hafler, Glatiramer
`acetate (Copaxone) induces degenerate, Th2-polarized immune responses
`in patients with multiple sclerosis, J. Clin. Invest. 105 (2000) 967–976.
`[11] C. Farina, F.T. Bergh, H. Albrecht, E. Meinl, A. Yassouridis, O. Neuhaus,
`R. Hohlfeld, Treatment of multiple sclerosis with Copaxone (COP): Elispot
`assay detects COP-induced interleukina-4 and interferon-␥ response in
`blood cells, Brain 124 (2001) 705–719.
`[12] C. Farina, S. Wagenpfeil, R. Hohlfeld, Immunological assay for assessing
`the efficacy of glatiramer acetate (Copaxone) in multiple sclerosis: a pilot
`study, J. Neurol. 249 (2002) 1587–1592.
`[13] B. Gran, L.R. Tranquill, M. Chen, B. Bielekova, W. Zhou, S. Dhib-Jalbut,
`R. Martin, Mechanisms of immunomodulation by glatiramer acetate, Neu-
`rology 55 (2000) 1704–1714.
`[14] A. Hug, M. Korporal, I. Schr¨oder, J. Haas, K. Glatz, B. Storch-Hagenlocher,
`B. Wildeman, Thymic export function and T cell homeostasis in patients
`with relapsing-remitting multiple sclerosis, J. Immunol. 170 (2003)
`432–437.
`[15] N.J. Karandikar, M.P. Crawford, X. Yan, R.B. Ratts, J.M. Brenchley, D.R.
`Ambrozak, A.E. Lovett-Racke, E.M. Frohman, P. Satstny, D.C. Douek,
`R.A. Koup, M.K. Racke, Glatiramer acetate (Copaxone) therapy induces
`CD8+ T cell responses in patients with multiple sclerosis, J. Clin. Invest.
`109 (2002) 641–649.
`
`[16] A. Miller, S. Shapiro, R. Gershtein, A. Kinarty, H. Rawahdeh, S. Honigman,
`N. Lahat, Treatment of multiple sclerosis with Copolymer-1 (Copaxone):
`implicating mechanisms of Th1 to Th2/Th3 immune-deviation, J. Neu-
`roimmunol. 92 (1998) 113–121.
`[17] O. Neuhaus, C. Farina, H. Wekerle, R. Hohlfeld, Mechanisms of action
`of glatiramer acetate in multiple sclerosis, Neurology 56 (2001) 702–
`706.
`[18] O. Neuhaus, C. Farina, A. Yassouridis, H. Wiendl, F.T. Bergh, T. Dose,
`H. Wekerle, R. Hohlfeld, Multiple sclerosis: comparison of copolymer-1-
`reactive T cell lines from treated and untreated subjects reveals cytokine
`shift from T helper 1 to T helper 2 cells, Proc. Natl. Acad. Sci. U.S.A. 97
`(2000) 7452–7457.
`[19] C.M. Poser, D.W. Paty, L. Scheinberg, W.I. McDonald, F.A. Davis, G.C.
`Ebers, K.P. Johnson, W.A. Sibley, D.H. Silberger, W.W. Tourtellote, New
`diagnostic criteria for multiple sclerosis: guidelines for research protocols,
`Ann. Neurol. 13 (1983) 227–231.
`[20] J.W. Rose, H.E. Watt, A.T. White, N.G. Carlson, Treatment of multiple
`sclerosis with an anti-interleukin-2 receptor monoclonal antibody, Ann.
`Neurol. 56 (2004) 864–867.
`[21] P. Saule, J. Trauet, V. Dutriez, V. Lekeux, J.P. Dessaint, M. Labalette, Accu-
`mulation of memory T cells from childhood to old age: central and effector
`memory cells in CD4+ versus effector memory and terminally differenti-
`ated memory cells in CD8+ compartment, Mech. Age. Dev. 127 (2006)
`274–281.
`[22] C. Weder, G.M. Baltariu, K.A. Wyler, H.J. Gober, C. Lienert, M. Schluep,
`E.W. Rad¨u, G. De Libero, L. Kappos, P.W. Duda, Clinical and immune
`responses correlate in glatiramer acetate therapy of multiple sclerosis, Eur.
`J. Neurol. 12 (2005) 869–878.
`[23] E. Wiesemann, J. Klatt, D. S¨onmez, R. Blasczyk, F. Heidenreich, A. Wind-
`hagen, Glatiramer acetate (GA) induces IL-13/IL-5 secretion in na¨ıve T
`cells, J. Neuroimmunol. 119 (2001) 137–144.
`[24] V.W. Yong, Differential mechanisms of action of interferon-␤ and glati-
`ramer acetate in MS, Neurology 59 (2002) 802–808.
`[25] T. Ziemssen, T. Kumpfel, W.E.F. Klinker, O. Neuhaus, R. Hohlfeld, Glati-
`ramer acetate-specific T-helper 1 and 2-type cell lines produce BDNF:
`implications for multiple sclerosis therapy, Brain 125 (2002) 2381–2391.
`
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`YEDA EXHIBIT NO. 2125
`MYLAN PHARM. v YEDA
`IPR2015-00644