- Email: [email protected]
Anti-inflammatory and immunomodulatory potential of human immunoglobulin applied intrathecally in Lewis rat experimental autoimmune neuritis
Accepted Manuscript Anti-inflammatory and immunomodulatory potential of human immunoglobulin applied intrathecally in Lewis rat experimental autoimmune neuritis
Kalliopi Pitarokoili, Felix Kohle, Jeremias Motte, Oluwaseun Fatoba, Xiomara Pedreituria, Ralf Gold, Min-Suk Yoon PII: DOI: Reference:
S0165-5728(17)30063-2 doi: 10.1016/j.jneuroim.2017.05.008 JNI 476570
To appear in:
Journal of Neuroimmunology
Received date: Revised date: Accepted date:
14 February 2017 14 May 2017 15 May 2017
Please cite this article as: Kalliopi Pitarokoili, Felix Kohle, Jeremias Motte, Oluwaseun Fatoba, Xiomara Pedreituria, Ralf Gold, Min-Suk Yoon , Anti-inflammatory and immunomodulatory potential of human immunoglobulin applied intrathecally in Lewis rat experimental autoimmune neuritis, Journal of Neuroimmunology (2017), doi: 10.1016/ j.jneuroim.2017.05.008
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ACCEPTED MANUSCRIPT Title:
Anti-inflammatory and immunomodulatory potential of human immunoglobulin applied intrathecally in Lewis rat experimental autoimmune neuritis
Pedreituria (1), Ralf Gold (1), Min-Suk Yoon (1) (1)
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Kalliopi Pitarokoili (1), Felix Kohle (1), Jeremias Motte (1), Oluwaseun Fatoba (1), Xiomara
Department of Neurology, St. Josef Hospital, Ruhr-University of Bochum, Germany
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Correspondence to: Kalliopi Pitarokoili, MD, MSc
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Dept. of Neurology, Ruhr University, St. Josef-Hospital, Gudrunstr. 56, 44791 Bochum, Germany.
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Email: [email protected]
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Tel: 0049-17662923235, Fax: 0049-234 5093024
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ACCEPTED MANUSCRIPT Abstract
Intravenous human immunoglobulins dominate in the treatment of autoimmune neuropathies. We introduce intrathecal application as a new option for experimental autoimmune neuritis in Lewis rats. After immunisation with neuritogenic P2 peptide, we show a therapeutic and
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preventive effect of intrathecal human immunoglobulins (5-40 mg/kg) on clinical and electrophysiological neuritis signs. Histology corroborated a lower degree of inflammation, demyelination, ICAM-1-dependent blood-nerve-barrier permeability and complement activation in the sciatic nerve. After preventive application, immunoglobulins induced a Th2
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cytokine shift in the peripheral nerves already before clinical neuritis signs. Intrathecal immunoglobulin application could be a novel immunomodulatory option for autoimmune
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neuropathies.
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Highlights
Intrathecal immunoglobulins (Ig) access the main site of inflammation in EAN
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Intrathecal Ig ameliorates clinical signs in Lewis rats neuritis model
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It reduces autoimmune inflammation, demyelination and complement activation In a preventive concent at low concentrations it induces a Th2 cytokine shift
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Intrathecal Ig could be a novel treatment option for autoimmune neuropathies
Keywords: autoimmune neuropathies ; immunoglobulins ; experimental autoimmune neuritis ; autoimmunity ; inflammation ; immunotherapy
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Abbreviations: AUC = Area under curve CFA = complete Freund`s adjuvant
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CIDP = chronic inflammatory demyelinating polyneuropathy CMAP = compound muscle potentials DAPI = 4`,6-Diamidin-2-phenylindol EAN = experimental autoimmune neuritis
ICAM1 = intercellular adhesion molecule 1
i.p. = intraperitoneally
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IVIg = intravenous immunoglobulins
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IgG = immunoglobulin G
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GBS = Guillain-Barré syndrome
MNCV = motor nerve conduction velocity
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p.i. = post immunisation
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PBS = phosphate buffer saline
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PNS = peripheral nervous system
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1. Introduction
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The first use of subcutaneous human immunoglobulins dates back to 1952 for patients with agammaglobulinemia (Burton et al., 1952, Buckley et al., 1998). Intravenous human immunoglobulins (IVIg) were shown effective for idiopathic thrombocytopenic purpura in 1981 (Imbach et al., 1981). Since then, their application has been extended to many
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autoimmune disorders including diseases of the central and peripheral nervous system. Their immunomodulatory properties combined with an attractive safety profile are the main reasons
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for their establishment as a first-line treatment for autoimmune diseases of the peripheral nervous system (PNS). Patients with the acute form (Guillain-Barré Syndrom, GBS) or the
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chronic form of peripheral autoimmune neuritis (chronic inflammatory demyelinating polyradiculoneuropathy, CIDP) improve rapidly during the first 24-48h after intravenous
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application of IVIg and remain stable during the following weeks, as immunoglobulin G
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(IgG) has a long half-life of two to three weeks (Lünemann et al., 2015). Human IVIg formulations available in market consist of purified IgG products from pooled human plasma
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of up to 60.000 donors. The content of unmodified IgG is approximately 95 % with only trace amounts of immunoglobulin A or immunoglobulin M. Patients with GBS are treated with 0.4g/kg body weight daily for 5 days whereas patients with CIDP receive initially 2 g/kg body weight in 2-4 days followed by a maintenance treatment of 1 g/kg body weight every 3-4 weeks intravenously (Hughes et al., 2008). Taking into account the price per gram of IVIg (approximately $70), it is obvious that the overall cost of this treatment for CIDP patients is extremely high (Blackhouse et al., 2010). The optimal dosing schema has to be adjusted individually as clinical experience shows that the optimal combination of dosage and 4
ACCEPTED MANUSCRIPT frequency of application varies between subjects and during the course of disease (Yoon et al., 2011, Willison et al., 2016). Subcutaneous route of administration has been tested in neuritis patients in terms of safety, tolerability and patients preference, as a dose-saving option, however, the dosages required remain relatively high (0.2 g/kg - 0.4 g/kg twice a week) (Van Schaik et al., 2016).
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The immunomodulatory mechanisms of immunoglobulin in autoimmune PNS diseases have been widely investigated in human disease. Because of their polyvalent nature, a wide spectrum of immunomodulatory effects mostly in the inflamed PNS either through the F(ab´)2 or through the Fc segment has been shown. F(ab´)2 segment can block different receptors
and
neutralize
cytokines,
complement,
adhesion-molecules
and
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cellular
autoantibodies (Viard et al., 1998, Le Pottier et al., 2007, Schwab et al., 2013). Fc Segment
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can modulate activating (high-affinity FcγRIA, FcγRIIA/C, FcγRIIIA/B, FcγRIV) and inhibitory (FcγRIIB) FcγR expression on immune cells (Samuelsson et al., 2001, Hansen et
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al., 2003, Anthony et al., 2011). These mechanisms have been widely investigated in the prototype model of animal autoimmune PNS disease, the experimental autoimmune neuritis
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(EAN), revealing a complex and not yet fully understood immunomodulatory effect (Enders
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et al., 1997, Gabriel et al., 1997, Lin at al., 2007, Niknami et al., 2013, Kajii et al., 2014). In view of this complexity, it has to be pointed out that the influence of each these pathways
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from IVIg, depends not only on the particular autoimmune disease but also on the route of administration.
In this study, we describe the immunomodulatory effects of human immunoglobulin (hIg) applied intrathecally in the Lewis rat model of EAN. This route of administration has gained a preference for multiple diseases with central (spinal) nervous system involvement such as baclofen application against spasticity or chemotherapy in leptomeningeal neoplastic involvement (Otero-Romero et al., 2016, Mack at al., 2016). Intrathecal opioid injection can modulate central/spinal sensitisation in chronic regional pain syndromes by bypassing the 5
ACCEPTED MANUSCRIPT blood-brain barrier and providing direct accessibility to central and peripheral neural structures (Pope at al., 2016). Furthermore, an increasing number of publications have reported the intrathecal use of monoclonal antibodies in multiple sclerosis (Studer et al., 2014, Bonnan et al., 2014, Tomita et al., 2014, Doorduijn et al., 2016). We present the first report of a new effective and dose-saving route of administration of immunoglobulins for dysimmune
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neuropathies.
2. Materials and methods
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2.1. Antigens
The neuritogenic P2 peptide, corresponding the amino acids 53-78 of rat myelin P2 protein,
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was synthesized by Dr. Rudolf Volkmer from Charité Universitätsmedizin (Berlin, Germany).
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2.2 Human immunoglobulin preparation
A human immunoglobulin preparation 10% Privigen® (CSL Behring) was used for all
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experiments. One ml of this preparation contains 100 mg of human immunoglobulin produced
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from the plasma of human donors (purity of at least 98% IgG). The distribution of the IgG subclasses is (approx. values): IgG1 67.8%, IgG2 28.7%, IgG3 2.3%, IgG4 1.2%. The
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maximum IgA content is 25 μg/ml. It contains approximately 250 mmol/L (range: 210 to 290) of L-proline.
2.3. Disease induction and clinical score assessment A total of 48 female Lewis rats were randomised for the therapeutic concept and a total of 59 rats were randomised for the preventive concept as described in the following section.The rats were 6–8 weeks old, they were purchased from Charles River Co. (Sulzfeld, Germany) and weighed 160–180 g when used for the following experiments. They were anesthetized by 1.5
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ACCEPTED MANUSCRIPT % - 2.0 % halothane in oxygen. They were immunized by subcutaneous injection of 250 µg P253-78 peptide in phosphate buffered saline (PBS) into the root of the tail, emulsified in an equal volume of complete Freund´s adjuvant (CFA) containing Mycobacterium tuberculosis H37RA (Difco, Detroit, MI, USA). Animals were weighted and scored for disease severity daily by two independent, blinded investigators. Disease severity was assessed clinically
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employing a scale ranging from zero to 10 originally described by Enders et al. (1998): 0 normal; 1 less lively; 2 impaired righting/limb tail; 3 absent righting; 4 atactic gait, abnormal position; 5 mild paraparesis; 6 moderate paraparesis; 7 severe paraplegia; 8 tetraparesis; 9 moribund; 10 death. All experiments were reviewed and approved by the North-Rhine-
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Westphalia, Germany authorities for animal experimentation (TVA 84-02.04.2014-A388).
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2.4. Intrathecal treatment with human immunoglobulins and dosage rationale The animals were anaesthetized by 1.5 % - 2.0 % halothane in oxygen and human
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immunoglobulins were injected intrathecally slowly within 4 seconds with a microsyringe in the following concentrations: for the preventive concept 5 mg/kg (n=4), 10 mg/kg (n=14), 20
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mg/kg (n=11) and 40 mg/kg (n=4) once daily on days 0, 2 and 4 post immunisation (p.i.), and
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for the therapeutic concept 5mg/kg, 10 mg/kg and 20 mg/kg once daily (n=12/group) on days 11, 13 and 15 p.i. in a volume of 40 µl immunoglobulin suspension. As control NaCl 0.9 % in
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a volume of 40 µl was used (n=12). The injection was performed with a 30G needle into the 4 to 5 lumbar intervertebral space and the correct place of the injection was confirmed by a movement of the tail (´tail flick´) as described by Fairbanks and coleagues (Fairbanks et al., 2003). The experiments were repeated twice or three times as stated. The dosages described above were calculated according to the dose reduction factor used for other antibodies, which are applied both intravenously and intrathecally. According to Perez and colleagues the dosage of Rituximab injected intrathecally is 0.14 mg/kg in humans whereas the common concentration used intravenously is 7 mg/kg (500 mg for an average of 7
ACCEPTED MANUSCRIPT 70kg person) (Perez et al., 2008). Therefore the dose reduction factor is about 50. The common concentration of intravenous immunoglobulins used in EAN is 400 mg/kg (Gabriel et al., 1997, Enders et al., 1997) so we used an approximate dose reduction factor of 20 (20 mg/kg) to 80 (5 mg/kg) in our experiments.
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2.5. Electrophysiological analysis
Nerve conduction tests were performed by a blinded investigator on the day before immunisation (-1) and on day 18 (maximum of natural disease course) p.i. The rats were anesthetized intraperitoneally (i.p.) with xylazine and ketamine (10 mg/kg and 50 mg/kg
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respectively). Using a fully digital recording Keypoint apparatus (Dantec, Skovlunde, Denmark) and paired needle electrodes inserted into the sciatic notch (hip; proximal) or the
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popliteal fossa (distal), the sciatic nerve was stimulated with supramaximal rectangular pulses of 0.05 ms duration and the resulting compound muscle action potential (cMAP) was
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recorded from needle electrodes placed subcutaneously over the dorsal foot muscles. A ground electrode was placed between the distal stimulating electrode and the active recording
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electrode. To calculate the motor nerve conduction velocity (mNCV), the distance between
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stimulating cathodes was divided by the difference of the latency. Similarly, the persistence and minimum latency of 10 F-waves evoked by stimulation at the popliteal fossa were
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recorded for the right side (Tuck et al., 1982, Taylor et al., 2003). Temperature differences were minimized by conducting the study as soon as the anesthesia had taken effect and by warming the leg with a heating lamp.
2.6. Histopathological assessment and immunohistochemistry After transcardial perfusion with PBS (Gibco) on day 18 or day 19 p.i. the two sciatic nerves were dissected and their segments were embedded in Tissue-Tek OCT Compound and snapfrozen in liquid nitrogen. For histopathology assessment, rat tissue was sectioned (8 µm) on a 8
ACCEPTED MANUSCRIPT cryostat (Leica Biosystems) and mounted on glass slides (Hartenstein, Würzburg, Germany). For the immunohistochemical staining, cryostat sections were fixated in acetone at 20°C for 10 minutes and were exposed to the mouse monoclonal antibodies (mAb) anti-rat 15-6A1 (Hycultec, Pan T-Cells CD3 1:100) and anti-rat ED1 (Hycultec, anti-CD68, macrophages, 1:100) using the avidin-biotin technique (Dako ARK KIT for Mouse Primary AB). The
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omission of the primary antibodies served as negative control. Specificity of the staining was also controlled on sections of peripheral lymphoid organs. The numbers of positive cells were counted at ×40 magnification for 12 sections per animal. The average results are expressed as cells per mm2 tissue section.
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For immunohistochemical stainings, cryostat sections were fixated in acetone at 20°C for 10 minutes and were exposed to the following monoclonal antibodies (mAb) anti-ICAM-1
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(1:100, Abcam, ab127160), anti-C3 (1:100, Abcam, ab11887), claudin-5 (1:100, Thermo Fisher Scientific, 4C3C2).
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Secondary antibodies conjugated with Alexa 555 (1:1000) or Alexa 488 (1:1000)
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(ThermoFisher, Schwerte, Germany) were used according to manufacturer’s protocol and DAPI (4',6' diamino-2-phenylindole·2HCl, Biozol, Eching, Germany) was used for
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fluorescent staining of DNA. Fluorescent signals were detected using an inverted fluorescence
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microscope (BX51; Olympus, Tokyo, Japan) equipped with an Olympus DP50 digital camera. For assessment of demyelination, lesions were identified by the accumulation of nuclei and absence of FluoroMyelinTM Red fluorescent stain (1:300, Molecular Probes) performed according to the manufacturer’s protocol. Images (20x-magnification) of twelve transverse sections of the sciatic nerve from each animal were digitally generated (Cell F 5.1, Olympus, Tokyo, Japan). The percentage of the area of demyelination per section was determined using image analysis software (ImageJ, National institutes of Health, Bethesda, USA). For histopathological assessment and immunohistochemistry, slides were blinded by a notinvolved third person and labelled with a numeric-code, which was unblinded after analysis. 9
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2.7. Isolation of mononuclear cells from lymph nodes and FACS analyses The inguinal lymph nodes were removed after transcardial perfusion with PBS (Gibco) on day 10 p.i. for the preventive setting under aseptic conditions. Single cell suspensions of mononuclear cells (MNC) from individual rats were prepared separately (preventive setting
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n=4/group). We evaluated the frequency of CD4+ T cells, CD11b+ cells, CD4+CD11b+ dendritic cells (DCs), CD4+ CD25+ FoxP3+ regulatory T-cells (Tregs) and CD4+CD11bMHCII+ plasmacytoid DCs by fluorescence-activated cell sorting (FACS) staining (eBioscience, San Diego, CA). FACS analyses were performed with a FACS Canto II (BD
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Pharmingen, Heidelberg, Germany) machine and FlowJo software (Tree Star). Monoclonal antibodies purchased from BD Pharmingen or eBioscience were used to detect CD4, CD11b,
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CD25 and MHC-II in accordance with the manufacturer´s instructions. Intracellular staining for Foxp3 was performed using the Foxp3 Staining Set (eBioscience) according to the
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manufacturer’s instructions.
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2.8 Tissue preparation, RNA isolation and gene expression analyses with quantitative rt-PCR
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Total RNA was isolated from the sciatic nerve samples of rats 10 days p.i. (n=8) using the RNeasy Mini extraction kit (Qiagen, Hilden, Germany). All samples were treated with the
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RNA Stabilization Reagent (RNAlater, Qiagen, Hilden, Germany) at 37 °C overnight and stored at −80 °C until use. Total RNA was reverse-transcribed into cDNA according to the manufacturer's protocol for the Reverse Transcription System (Promega, Madison, WI). IFNgamma, TNF-alpha, IL-10 and IL-4 mRNA expression levels were analyzed by quantitative RT-PCR according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). Sequence-specific primers and probes were designed using pre-developed TaqMan® assay reagents (Applied Biosystems). rt-PCR amplifications were carried out using the real-time PCR System 7500 (Applied Biosystems). In brief, real-time PCR for RNA-to-CTTM 1-Step 10
ACCEPTED MANUSCRIPT was performed according to the following amplification protocol: 25°C for 5 min, 42°C for 30 min and 82°C for 5min (for cDNA synthesis), 95°C for 3 min (transcriptase inactivation), followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. β-Actin was used to normalize relative mRNA expression. To compare each mRNA and housekeeping β-Actin mRNA expression, the relative expression of PCR products was determined using the ΔΔCt method
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(Schmittgen and Livak, 2008). Each experiment was performed in duplicate and the mean Ct was used in the equation.
2.9. Statistical methods
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Statistical analyses were performed by GraphPad Prism 6 software (GraphPad Software Inc., San Diego, USA). Data are provided as mean ± SEM (standard error of mean). Differences
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between pairs of groups were tested by Student’s t-test. Differences between three or more groups were tested by one-factor analysis of variance (ANOVA). Area under the curve (AUC)
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was calculated for clinical courses and analysed by one-way analysis of variance (ANOVA)
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combined with Tukey`s multiple comparison test. In all experiments, a P value of < 0.05 was
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significant.
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defined as statistically significant, and P < 0.0001 was considered as highly statistically
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ACCEPTED MANUSCRIPT 3. Results
3.1. Intrathecal human immunoglobulins applied in a therapeutic concept attenuate the clinical course of experimental autoimmune neuritis After immunisation with P2 protein peptide 53-78, clinical signs of EAN begun around day
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10 p.i. At days 11, 13 and 15 p. i. 0.9% NaCl or human immunoglobulins were applied intrathecally. The incidence of EAN for the control group was 100 % and the groups receiving human immunoglobulins showed an incidence of 100 % (5 mg/kg), 75 % (10 mg/kg) or 66 % (20 mg/kg) respectively. A dose-dependent amelioration of EAN clinical
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signs was documented (AUC, NaCl-treated vs. 20 mg/kg hIgs ***p<0.0001) (Figure 1). There was a non-significant reduction of body weight of sham-treated controls during the
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course of EAN as compared to rats treated with hIgs, in which body weight remained constant
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(data not shown).
3.2. Intrathecal human immunoglobulins improve mostly proximal but also distal nerve
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conduction
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To elucidate the effect of intrathecal immunoglobulins regarding proximal and distal demyelination, we performed electrophysiological measurements of the sciatic nerve at the
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maximum of the clinical course (day 18 p.i.) as described in material and methods. The following measures were evaluated: 1) spinal root involvement (proximal demyelination), depicted by F-wave persistence and F-wave latencies and 2) degree of distal demyelination, as implied by the reduction of the motor nerve conduction velocity (mNCV). Persistence of F-wave response, as defined by the number of F-waves elicited after 10 distal stimulations, was 40 % at the peak of disease (day 18 p.i.) for the NaCl-treated rats and 50 % for the 5 mg/kg intrathecal Igs-treated group (non-significant compared to control group). On the contrary, persistence of F-wave response was 95 % for the 10 mg/kg and 20 mg/kg 12
ACCEPTED MANUSCRIPT intrathecal Igs-treated groups (***p< 0.0001 compared to control group) consistent with early proximal sciatic nerve involvement in control animals and indicating the crucial protective role of immunoglobulins injected intrathecally against inflammatory root involvement (Figure 2A, B). The average minimum latencies of the elicitable F-waves were slightly prolonged in NaCl-
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treated group and in 5 mg/kg immunoglobulins-treated group between the peak of disease (day 18 p.i.) and day -1 p.i., whereas there was no difference for the 10 mg/kg hIg-treated group and the 20 mg/kg hIg-treated-group (data not shown).
mNCV was significantly reduced in the NaCl-treated rats (mean mNCV on day 18 p.i. 29, 7
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m/s vs. day -1 p.i. 50.4 m/s, ****p < 0.0001, n=4), whereas no difference of the mean mNCV on day -1 was seen for hIg-treated groups (Figure 2C), indicating the additional protective
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role of intrathecal application of immunoglobulin against distal demyelination.
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3.3. Intrathecal human immunoglobulin reduces histological signs of demyelination and T cell and macrophage infiltration in the sciatic nerve
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Next, we analyzed if the electrophysiological reduction of proximal and distal demyelination
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correlates with histological signs of myelination. Figure 3 shows representative pictures of FluoroMyelinTM staining. Administration of 10 and 20 mg/kg intrathecal immunoglobulins
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significantly reduced the percentage of demyelination of the sciatic nerves when compared to NaCl-treated control rats (***p<0.0001, n=4, Figure 3). Next, we showed that clinical improvement correlates with the reduction of inflammatory infiltration of the PNS. Histological data showing inflammation within the PNS are depicted in Figure 4. Intrathecal administration of 20 mg/kg immunoglobulins on days 11, 13 and 15 p.i. significantly reduced infiltration of macrophages and lymphocytes in the sciatic nerves compared to NaCl-treated control group, when examined on day 18 p.i. (***p<0.0001). Lower concentrations of immunoglobulins (5 and 10 mg/kg) showed a less pronounced 13
ACCEPTED MANUSCRIPT reduction of inflammatory cells (Figure 4). Interestingly, inflammatory infiltrates were reduced both in the distal and proximal parts of the sciatic nerves (data not shown).
3.4.
Intrathecal
immunoglobulins
preserve
ICAM-1-dependent
blood-nerve-barrier
permeability and reduce C3 expression in peripheral nerves
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Thereafter, we surveyed possible immunological mechanisms for the proximal but also distal reduction of infiltrating inflammatory cells in the peripheral nerves after intrathecal immunoglobulin treatment.
Among the multiple reported effects of immunoglobulins applied systemically are the
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reduction of endothelial transmigration molecules such as ICAM-1 (intercellular adhesion molecule). and of the C3 complement fragment (Basta et al., 1989).
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ICAM-1 expression was significantly reduced for 20 mg/kg-treated rats correlating with the most prominent reduction of inflammatory infiltrates for this concentration (***p<0.0001,
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Figure 5). In order to distinguish blood nerve preservation effects of Ig on the blood nerve barrier, which are unrelated to inflammatory infiltration we analysed histological claudin-5
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treatment (Figure 6).
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expression in the peripheral nerves, which showed no significant differences after Ig
Finally, we analysed C3 fragment expression in the sciatic nerves at the peak of disease, was
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significantly reduced after intrathecal immunoglobulin expression (***p<0.0001, Figure 7).
3.5. Preventive intrathecal immunoglobulin treatment reduced clinical, electrophysiological and histological EAN signs Therapeutic intervention with intrathecal immunoglobulins showed a remarkable effect in clinical, electrophysiological and histological signs of neuritis. The question arises if preventive intrathecal immunoglobulin injection at the EAN induction phase (days 0, 2, 4 p.i.) ameliorates clinical, electrophysiological and histological signs of dysimmune neuritis. As 14
ACCEPTED MANUSCRIPT shown in Figure 8 intrathecal immunoglobulins reduced EAN but their effect was more prominent for the low concentrations of 5 and 10 mg/kg (*p< 0.05). This effect was confirmed in electrophysiological and histological terms as only the lower concentrations of intrathecal immunoglobulins showed a preservation of mNCV on day 19 p.i. and a reduction
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of the inflammatory infiltrates (Figure 8 B, C, D).
3.6. Intrathecal immunoglobulins in a preventive concept reduce early C3 expression (day 10 p.i.) and induce an early Th2 cytokine shift in peripheral nerves
We showed above that immunoglobulins applied intrathecally in a therapeutic concept induce
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a dose-dependent reduction of C3 fragment expression and a preservation of blood-nervebarrier at disease maximum.
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In the preventive concept, the lowest concentration of hIg showed the most prominent effect implying possibly different mechanisms of action in this EAN phase. We proceeded to test
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the mechanisms of action of intrathecal immunoglobulins applied in the induction EAN phase by analyzing histological C3 fragment and ICAM expression as well as Th1 and Th2 cytokine
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profiles at day 10 p.i. (before clinical neuritis signs).
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At day 10 p.i. there was no statistically significant difference in the inflammatory infiltrates, ICAM or claudin-5 expression between NaCl-treated and hIg treated rats (data not shown).
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However, in the rats treated with a lower concentration of human immunoglobulins a statistically significant reduction of C3 histological expression in sciatic nerves was still induced just before disease symptoms started (Figure 9A). Furthermore, a Th2 cytokine shift was observed for the lower concentration of 5 mg/kg at that time point, as only IL-10 and IL4 mRNA expression showed a statistically significant increase before disease initiation (Figure 9 B, C, D, E). In order to detect possible peripheral effects of intrathecally applied immunoglobulins, we also isolated periherally mononuclear cells from the inguinal lymph nodes at day 10 p.i. and 15
ACCEPTED MANUSCRIPT analyzed them with FACS analysis. We evaluated the frequency of CD4+ T cells, CD11b+ cells, CD4+CD11b+ dendritic cells (DCs), CD4+ CD25+ FoxP3+ regulatory T-cells (Tregs) and CD4+CD11b-MHCII+ plasmacytoid DCs. No statistically significant differences were found
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between immunoglobulin and NaCl-treated rats for all populations tested (data not shown).
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ACCEPTED MANUSCRIPT 4. Discussion
In the current study, we present a new perspective of the classical treatment of autoimmune neuropathies with immunoglobulins using the alternative route of intrathecal application. This application route has not yet been used for immunoglobulins in autoimmune diseases of the
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peripheral nervous system although the first sites of inflammation and increase of blood-nerve barrier permeability are the proximal nerve roots, as indicated by the early intrathecal protein increase found in these patients (Hughes et al., 2006, Kerasnoudis et al., 2014). The intrathecal injection of a 98% total IgG protein preparation in low concentrations exerted
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unique immunomodulatory and anti-inflammatory actions in our model.
In the therapeutic concept, intrathecal immunoglobulins achieve a dose-dependent
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improvement of clinical signs, which correlates with a histological reduction of inflammatory infiltrates in the sciatic nerves.
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Firstly, we investigated whether this anti-inflammatory effect of intrathecal immunoglobulins is focused near the injection site (close to the in the nerve roots) or rather distally.
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Electrophysiologically, we found a prominent preservation of F-wave persistence as an
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indicator of an improvement of proximal demyelination as well as an unchanged motor conduction velocity as a sign of a distal preservation of myelination. These findings were
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histologically confirmed by large-field immunostaining for intact myelin, yet a thorough histological analysis using electron microscopy was not performed. We conclude that intrathecal injection in the effector phase reduces inflammatory activity beginning at the injections sites and extending into the whole nerve thereby achieving sufficient improvement of electrophysiological signs of demyelination. In order to dissect possible mechanisms of action of the local intrathecal injection of immunoglobulins, we analyzed possible immunomodulatory mechanisms in the peripheral nervous system reported before for systemically applied immunoglobulins. 17
ACCEPTED MANUSCRIPT Firstly, we showed a local reduction of C3 fragment expression in the peripheral nerves at the maximum of disease. This effect has been already described for systemically applied immunoglobulins and has been attributed to the F(ab')2 region of IgG. This region is shown to neutralize activated complement proteins, including C3a and inhibit the uptake of C3b and C4b onto the cell surface, thereby preventing complement-mediated tissue damage (Basta et
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al., 1989). However, for the intravenous application of human immunoglobulins, high concentrations are needed to exert these peripheral and local anti-inflammatory actions. In our model the disrupted blood-nerve-barrier during inflammation in combination with the local injection of immunoglobulins enables low concentrations of immunoglobulins to exert these
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anti-inflammatory actions.
Furthermore, the reduction of inflammatory infiltrates correlated with the downregulation in
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the histological expression of ICAM molecule in the peripheral nerves. This effect has been reported before for patients with autoimmune dermatomyositis in muscle biopsies after
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systemically immunoglobulin treatment (Rigal et al., 1994) as well as in sera of GBS patients after IVIg infusion (Sharief et al., 1999). Dermatomyositis studies revealed a downregulation
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of ICAM gene expression in treatment-responders was found (Raji et al., 2005). As described
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later in the discussion and based on our results this effect on ICAM-1 is secondary to the mononuclear cell infiltration.
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To our knowledge no reports exist on the effects of Ig on molecules preserving the bloodnerve barrier during inflammation such as claudin-5. However, there are reports which describe a decrease of claudin-5 in the sural nerve of CIDP patients (Kanda et al., 2004, Shimizu et al., 2014). In our model, there was no influence on the histological expression of claudin-5 after Ig treatment neither on the therapeutic concept nor at the preventive concept, therefore we conclude that the main effects of intrathecal Ig treatment in our case rather stem from immunomodulatory/anti-inflammatory effects than from a direct effect on blood-nerve barrier permeability. 18
ACCEPTED MANUSCRIPT Finally, further aspects of local (intrathecal) immunomodulatory action of immunoglobulins on cellular inflammatory receptors (FasL-Fas), autoantibodies or Fc-gamma-receptors could surely play a role in our model. This fact points out the great potential of this application route in inflammatory neuropathies and has to be investigated in the future. One aspect of immunoglobulin treatment, which is highly relevant for further clinical
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application, is the correlation of dose and time-point of application with the clinical symptoms of neuritis. These aspects are nowadays individually adjusted for each patient in order to achieve clinical stability. The fact that immunoglobulins have an anti-inflammatory action at clinical CIDP relapse or in the acute GBS phase is well known. Whether they also have an
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immunomodulatory effect on the events which contribute to chronic autoimmune inflammation, with immunoglobulins finally leading to prevention of further relapses is not
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known. This question was investigated in our model by applying immunoglobulins in a preventive concept combined with immunisation.
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We show that the effects of immunoglobulins in this phase are still present but surprisingly not in a dose-dependent manner. Low concentrations of immunoglobulins applied
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findings.
By
examining
the
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improvement
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intrathecally are sufficient to attenuate clinical EAN signs, which correlated with an
immunomodulatory effects in the peripheral nerves at day 10 p.i. we were able to dissect the
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primary effects of immunoglobulins on ICAM-1 and C3 fragment without the secondary effects through the reduction of inflammatory infiltrates. Based on our findings, we conclude that immunoglobulins exert a direct effect on C3 fragment expression but probably only a secondary effect on ICAM-1 expression. The shift to Th2 cytokines already at this early time point emphasizes the preventive potential of immunoglobulins through a possible direct effect on cellular inflammatory receptors in the induction phase. The reason for this non-dose dependent effect in this phase could be a receptor-saturation-mediated effect in the noninflamed PNS at this early time point. Our findings imply that intrathecal immunoglobulins 19
ACCEPTED MANUSCRIPT could play a useful preventive role in autoimmune relapses but most importantly in lower concentrations. Concluding, we present a new perspective in immunoglobulin treatment in peripheral neuropathies by applying intrathecal immunoglobulin in a preventive and therapeutic concept. A transfer of our application model in human subjects could have many pitfalls, which were
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not obvious in the animal model. Firstly, the aspect of toxicity due to the high viscosity of immunoglobulin preparation and the existence of L-proline has to be taken into account. This can be solved by modifying these parameters in the immunoglobulin preparation. Furthermore, allergic reactions, which are described after intravenous infusion could be more
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pronounced after intrathecal injection. However, we believe that in the light of our current
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treatment option for neuritis patients.
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observations intrathecal application of immunoglobulin preparations could be a promising
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Acknowledgments - Conflicts of Interest and Source of Funding
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This study was supported by a scientific grant from CSL Behring.
Kalliopi Pitarokoili has received travel grants from Biogen Idec and Bayer Pharmaceuticals and speakers honoraria from Biogen Idec, Novartis and Bayer Pharmaceuticals. Felix Kohle, Oluwaseun Fatoba and Xiomara Pedreituria report no disclosures. Jeremias Motte received travel grants from Biogen Idec. R. Gold has received consultation fees and speakers honoraria from Bayer Schering, Biogen Idec, Merck Serono, Novartis, Sanofi-Aventis and TEVA. He also acknowledges grant support from Bayer Schering, Biogen Idec, Merck-Serono, Sanofi-
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ACCEPTED MANUSCRIPT Aventis and TEVA. Min-Suk Yoon received consulting, speaker honoraria and scientific grant from CSL Behring, speaker honoraria from Grifols.
Figure Legends: Figure 1: Representative clinical course of EAN rats (n=4). EAN was induced in Lewis rats
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by immunisation on day 0 with P2 peptide 53-78 plus CFA. Rats received human immunoglobulins (hIgs) intrathecally at doses of 5 mg/kg, 10 mg/kg and 20 mg/kg on days 11,13 and 15 post immunisation. Control rats received NaCl 0.9% only. The experiment was repeated three times, each time with n=4/group. Mean values and SEM are depicted.
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Statistical analysis and p-value calculation were performed calculating the area under the curve. Comparisson of AUC was done using one-way ANOVA combined with Tukey`s
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multiple comparisons test. NaCl treated vs 20 mg/kg ***p< 0.0001.
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Figure 2: Representative traces of F waves at disease maximum (day 18 p.i.) from NaCltreated rats (A) and 20 mg/kg intrathecal Igs-treated rats (B). NaCl treated rats presented with
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a reduced persistence of F-wave response whereas Ig-treated rats showed no a normal
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response indicating the effect of effect of Igs in reducing proximal demyelination. C. Motor nerve conduction velocity of the sciatic nerve one day before immunisation (d-1) and at the
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peak of disease (day 18 p.i.) (n=4/group): After proximal and distal stimulation of the sciatic nerve the conduction velocity was calculated. A statistical significant reduction of the MNCV appeared only for the NaCl-treated group (n=4) but no difference in the mNCV was seen for all immunoglobulin-treated groups, indicating a protective role of immunoglobulins against distal demyelination. Mean values and SEM are depicted, p-values ****p< 0.0001. The experiments were repeated three times with n=4/group.
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ACCEPTED MANUSCRIPT Figure 3: Representative pictures and statistical analysis of Fluoromyelin staining for sciatic nerve transverse sections of rats (n=4/group) treated with immunoglobulins 5, 10 and 20 mg/kg and NaCl-treated animals, showing massive demyelination for NaCl-treated rats and preservation of myelin for Igs treated groups (***p<0.0001). The experiment was repeated
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three times with similar results. Scale bars indicate 100µm.
Figure 4: Mean numbers of A. CD68+ macrophages and B. T cells per mm2 sciatic nerve sections as measured by immunohistochemistry on day 18 p.i. from EAN rats (n=4/group)
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receiving intrathecal immunoglobulins in a therapeutic concept at different doses (5 mg/kg, 10 mg/kg, 20 mg/kg) and NaCl-treated rats at days 11, 13, 15 post immunisation. Mean
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three times with similar results.
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values and SEM are depicted, p-values***p<0.0001 *p<0.05. The experiment was repeated
Figure 5: Representative pictures and statistical analysis of ICAM staining for sciatic nerve
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transverse sections of rats (n=4/group) treated with immunoglobulins 5, 10 and 20 mg/kg and
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NaCl-treated animals, showing a reduction of ICAM histological expression for 10 and 20 mg/kg Ig-treated rats, which implies a protective secondary effect on blood-nerve-barrier
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permeability (***p<0.0001). The experiment was repeated three times with similar results. Scale bars indicate 100µm.
Figure 6: Representative pictures and statistical analysis of claudin-5 staining for sciatic nerve transverse sections of rats (n=4/group) treated with immunoglobulins 5, 10 and 20 mg/kg and NaCl-treated animals, showing a tendency towards no statistical significant difference in claudin-5 histological expression for Ig-treated rats. The experiment was repeated three times with similar results. Scale bars indicate 100µm. 22
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Figure 7: Representative pictures and statistical analysis of C3 staining for sciatic nerve transverse sections of rats (n=4/group) treated with immunoglobulins 5, 10 and 20 mg/kg and NaCl-treated animals, showing a reduction of C3 histological expression for 10 and 20 mg/kg Ig-treated rats (***p<0.0001). The experiment was repeated twice with similar results. Scale
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bars indicate 100µm.
Figure 8: A. Clinical course of EAN in Lewis rats after intrathecal immunoglobulins in a preventive concept. EAN was induced in Lewis rats by immunisation on day 0 with P2
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peptide 53-78 plus CFA. Rats received human immunoglobulins (hIgs) intrathecally at doses of 5 mg/kg, 10 mg/kg and 20 mg/kg on days 0, 2 and 4 post immunisation. Control rats
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received NaCl 0.9% only. Pooled results from three independent experiments (n=4-12/group). Mean values and SEM are depicted. AUC, NaCl treated vs 5 mg/kg *p< 0.05.
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B. Motor nerve conduction velocity of the sciatic nerve one day before immunisation (d-1) and at day 19 p.i. (n=4/group): After proximal and distal stimulation of the sciatic nerve the
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conduction velocity was calculated. A statistical significant reduction of the MNCV appeared
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only for the NaCl-treated group (n=4) but no difference in the mNCV was seen for all immunoglobulin-treated groups, indicating a protective role of immunoglobulins against
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distal demyelination. Mean values and SEM are depicted, p-values ***p< 0.0001. Pooled results from three independent experiments (n=4-12/group). C, D Mean numbers of C. CD68+ macrophages and D. T cells per mm2 sciatic nerve sections as measured by immunohistochemistry on day 19 p.i. from EAN rats (n=8/group) receiving intrathecal immunoglobulins in a therapeutic concept at different doses (5, 10 and 20 mg/kg) and NaCl-treated rats at days 0, 2, 4 post immunisation. Mean values and SEM are depicted, p-values****p<0.0001.
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ACCEPTED MANUSCRIPT Figure 9 A. C3 histological expression at day 10 p.i. for sciatic nerve transverse sections of rats (n=4/group) treated with immunoglobulins 5 and 40 mg/kg and NaCl-treated animals, showing a reduction of C3 histological expression only for 5 mg/kg hIg-treated rats (***p<0.0001, **p<0.001). Relative gene expression of Th1 cytokines (B. IFN-gamma,D. TNF-alpha) and Th2 cytokines
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(C. IL10, D. IL-4) in sciatic nerves at day 10 p.i. after application of 5 and 40 mg/kg hIgs showing a statitstical signifcant increase of IL-10 and IL-4 cytokines as a sign of a Th2
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cytokine shift.
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ACCEPTED MANUSCRIPT Highlights Intrathecal immunoglobulins (Ig) access the main site of inflammation in EAN Intrathecal Ig ameliorates clinical signs in Lewis rats neuritis model It reduces autoimmune inflammation, demyelination and complement activation In a preventive concent at low concentrations it induces a Th2 cytokine shift
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Intrathecal Ig could be a novel treatment option for autoimmune neuropathies
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Kalliopi Pitarokoili, Felix Kohle, Jeremias Motte, Oluwaseun Fatoba, Xiomara Pedreituria, Ralf Gold, Min-Suk Yoon PII: DOI: Reference:
S0165-5728(17)30063-2 doi: 10.1016/j.jneuroim.2017.05.008 JNI 476570
To appear in:
Journal of Neuroimmunology
Received date: Revised date: Accepted date:
14 February 2017 14 May 2017 15 May 2017
Please cite this article as: Kalliopi Pitarokoili, Felix Kohle, Jeremias Motte, Oluwaseun Fatoba, Xiomara Pedreituria, Ralf Gold, Min-Suk Yoon , Anti-inflammatory and immunomodulatory potential of human immunoglobulin applied intrathecally in Lewis rat experimental autoimmune neuritis, Journal of Neuroimmunology (2017), doi: 10.1016/ j.jneuroim.2017.05.008
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ACCEPTED MANUSCRIPT Title:
Anti-inflammatory and immunomodulatory potential of human immunoglobulin applied intrathecally in Lewis rat experimental autoimmune neuritis
Pedreituria (1), Ralf Gold (1), Min-Suk Yoon (1) (1)
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Kalliopi Pitarokoili (1), Felix Kohle (1), Jeremias Motte (1), Oluwaseun Fatoba (1), Xiomara
Department of Neurology, St. Josef Hospital, Ruhr-University of Bochum, Germany
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Correspondence to: Kalliopi Pitarokoili, MD, MSc
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Dept. of Neurology, Ruhr University, St. Josef-Hospital, Gudrunstr. 56, 44791 Bochum, Germany.
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Email: [email protected]
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Tel: 0049-17662923235, Fax: 0049-234 5093024
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ACCEPTED MANUSCRIPT Abstract
Intravenous human immunoglobulins dominate in the treatment of autoimmune neuropathies. We introduce intrathecal application as a new option for experimental autoimmune neuritis in Lewis rats. After immunisation with neuritogenic P2 peptide, we show a therapeutic and
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preventive effect of intrathecal human immunoglobulins (5-40 mg/kg) on clinical and electrophysiological neuritis signs. Histology corroborated a lower degree of inflammation, demyelination, ICAM-1-dependent blood-nerve-barrier permeability and complement activation in the sciatic nerve. After preventive application, immunoglobulins induced a Th2
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cytokine shift in the peripheral nerves already before clinical neuritis signs. Intrathecal immunoglobulin application could be a novel immunomodulatory option for autoimmune
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neuropathies.
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Highlights
Intrathecal immunoglobulins (Ig) access the main site of inflammation in EAN
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Intrathecal Ig ameliorates clinical signs in Lewis rats neuritis model
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It reduces autoimmune inflammation, demyelination and complement activation In a preventive concent at low concentrations it induces a Th2 cytokine shift
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Intrathecal Ig could be a novel treatment option for autoimmune neuropathies
Keywords: autoimmune neuropathies ; immunoglobulins ; experimental autoimmune neuritis ; autoimmunity ; inflammation ; immunotherapy
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Abbreviations: AUC = Area under curve CFA = complete Freund`s adjuvant
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CIDP = chronic inflammatory demyelinating polyneuropathy CMAP = compound muscle potentials DAPI = 4`,6-Diamidin-2-phenylindol EAN = experimental autoimmune neuritis
ICAM1 = intercellular adhesion molecule 1
i.p. = intraperitoneally
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IVIg = intravenous immunoglobulins
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IgG = immunoglobulin G
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GBS = Guillain-Barré syndrome
MNCV = motor nerve conduction velocity
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p.i. = post immunisation
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PBS = phosphate buffer saline
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PNS = peripheral nervous system
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1. Introduction
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The first use of subcutaneous human immunoglobulins dates back to 1952 for patients with agammaglobulinemia (Burton et al., 1952, Buckley et al., 1998). Intravenous human immunoglobulins (IVIg) were shown effective for idiopathic thrombocytopenic purpura in 1981 (Imbach et al., 1981). Since then, their application has been extended to many
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autoimmune disorders including diseases of the central and peripheral nervous system. Their immunomodulatory properties combined with an attractive safety profile are the main reasons
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for their establishment as a first-line treatment for autoimmune diseases of the peripheral nervous system (PNS). Patients with the acute form (Guillain-Barré Syndrom, GBS) or the
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chronic form of peripheral autoimmune neuritis (chronic inflammatory demyelinating polyradiculoneuropathy, CIDP) improve rapidly during the first 24-48h after intravenous
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application of IVIg and remain stable during the following weeks, as immunoglobulin G
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(IgG) has a long half-life of two to three weeks (Lünemann et al., 2015). Human IVIg formulations available in market consist of purified IgG products from pooled human plasma
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of up to 60.000 donors. The content of unmodified IgG is approximately 95 % with only trace amounts of immunoglobulin A or immunoglobulin M. Patients with GBS are treated with 0.4g/kg body weight daily for 5 days whereas patients with CIDP receive initially 2 g/kg body weight in 2-4 days followed by a maintenance treatment of 1 g/kg body weight every 3-4 weeks intravenously (Hughes et al., 2008). Taking into account the price per gram of IVIg (approximately $70), it is obvious that the overall cost of this treatment for CIDP patients is extremely high (Blackhouse et al., 2010). The optimal dosing schema has to be adjusted individually as clinical experience shows that the optimal combination of dosage and 4
ACCEPTED MANUSCRIPT frequency of application varies between subjects and during the course of disease (Yoon et al., 2011, Willison et al., 2016). Subcutaneous route of administration has been tested in neuritis patients in terms of safety, tolerability and patients preference, as a dose-saving option, however, the dosages required remain relatively high (0.2 g/kg - 0.4 g/kg twice a week) (Van Schaik et al., 2016).
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The immunomodulatory mechanisms of immunoglobulin in autoimmune PNS diseases have been widely investigated in human disease. Because of their polyvalent nature, a wide spectrum of immunomodulatory effects mostly in the inflamed PNS either through the F(ab´)2 or through the Fc segment has been shown. F(ab´)2 segment can block different receptors
and
neutralize
cytokines,
complement,
adhesion-molecules
and
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cellular
autoantibodies (Viard et al., 1998, Le Pottier et al., 2007, Schwab et al., 2013). Fc Segment
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can modulate activating (high-affinity FcγRIA, FcγRIIA/C, FcγRIIIA/B, FcγRIV) and inhibitory (FcγRIIB) FcγR expression on immune cells (Samuelsson et al., 2001, Hansen et
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al., 2003, Anthony et al., 2011). These mechanisms have been widely investigated in the prototype model of animal autoimmune PNS disease, the experimental autoimmune neuritis
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(EAN), revealing a complex and not yet fully understood immunomodulatory effect (Enders
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et al., 1997, Gabriel et al., 1997, Lin at al., 2007, Niknami et al., 2013, Kajii et al., 2014). In view of this complexity, it has to be pointed out that the influence of each these pathways
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from IVIg, depends not only on the particular autoimmune disease but also on the route of administration.
In this study, we describe the immunomodulatory effects of human immunoglobulin (hIg) applied intrathecally in the Lewis rat model of EAN. This route of administration has gained a preference for multiple diseases with central (spinal) nervous system involvement such as baclofen application against spasticity or chemotherapy in leptomeningeal neoplastic involvement (Otero-Romero et al., 2016, Mack at al., 2016). Intrathecal opioid injection can modulate central/spinal sensitisation in chronic regional pain syndromes by bypassing the 5
ACCEPTED MANUSCRIPT blood-brain barrier and providing direct accessibility to central and peripheral neural structures (Pope at al., 2016). Furthermore, an increasing number of publications have reported the intrathecal use of monoclonal antibodies in multiple sclerosis (Studer et al., 2014, Bonnan et al., 2014, Tomita et al., 2014, Doorduijn et al., 2016). We present the first report of a new effective and dose-saving route of administration of immunoglobulins for dysimmune
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neuropathies.
2. Materials and methods
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2.1. Antigens
The neuritogenic P2 peptide, corresponding the amino acids 53-78 of rat myelin P2 protein,
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was synthesized by Dr. Rudolf Volkmer from Charité Universitätsmedizin (Berlin, Germany).
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2.2 Human immunoglobulin preparation
A human immunoglobulin preparation 10% Privigen® (CSL Behring) was used for all
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experiments. One ml of this preparation contains 100 mg of human immunoglobulin produced
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from the plasma of human donors (purity of at least 98% IgG). The distribution of the IgG subclasses is (approx. values): IgG1 67.8%, IgG2 28.7%, IgG3 2.3%, IgG4 1.2%. The
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maximum IgA content is 25 μg/ml. It contains approximately 250 mmol/L (range: 210 to 290) of L-proline.
2.3. Disease induction and clinical score assessment A total of 48 female Lewis rats were randomised for the therapeutic concept and a total of 59 rats were randomised for the preventive concept as described in the following section.The rats were 6–8 weeks old, they were purchased from Charles River Co. (Sulzfeld, Germany) and weighed 160–180 g when used for the following experiments. They were anesthetized by 1.5
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ACCEPTED MANUSCRIPT % - 2.0 % halothane in oxygen. They were immunized by subcutaneous injection of 250 µg P253-78 peptide in phosphate buffered saline (PBS) into the root of the tail, emulsified in an equal volume of complete Freund´s adjuvant (CFA) containing Mycobacterium tuberculosis H37RA (Difco, Detroit, MI, USA). Animals were weighted and scored for disease severity daily by two independent, blinded investigators. Disease severity was assessed clinically
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employing a scale ranging from zero to 10 originally described by Enders et al. (1998): 0 normal; 1 less lively; 2 impaired righting/limb tail; 3 absent righting; 4 atactic gait, abnormal position; 5 mild paraparesis; 6 moderate paraparesis; 7 severe paraplegia; 8 tetraparesis; 9 moribund; 10 death. All experiments were reviewed and approved by the North-Rhine-
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Westphalia, Germany authorities for animal experimentation (TVA 84-02.04.2014-A388).
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2.4. Intrathecal treatment with human immunoglobulins and dosage rationale The animals were anaesthetized by 1.5 % - 2.0 % halothane in oxygen and human
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immunoglobulins were injected intrathecally slowly within 4 seconds with a microsyringe in the following concentrations: for the preventive concept 5 mg/kg (n=4), 10 mg/kg (n=14), 20
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mg/kg (n=11) and 40 mg/kg (n=4) once daily on days 0, 2 and 4 post immunisation (p.i.), and
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for the therapeutic concept 5mg/kg, 10 mg/kg and 20 mg/kg once daily (n=12/group) on days 11, 13 and 15 p.i. in a volume of 40 µl immunoglobulin suspension. As control NaCl 0.9 % in
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a volume of 40 µl was used (n=12). The injection was performed with a 30G needle into the 4 to 5 lumbar intervertebral space and the correct place of the injection was confirmed by a movement of the tail (´tail flick´) as described by Fairbanks and coleagues (Fairbanks et al., 2003). The experiments were repeated twice or three times as stated. The dosages described above were calculated according to the dose reduction factor used for other antibodies, which are applied both intravenously and intrathecally. According to Perez and colleagues the dosage of Rituximab injected intrathecally is 0.14 mg/kg in humans whereas the common concentration used intravenously is 7 mg/kg (500 mg for an average of 7
ACCEPTED MANUSCRIPT 70kg person) (Perez et al., 2008). Therefore the dose reduction factor is about 50. The common concentration of intravenous immunoglobulins used in EAN is 400 mg/kg (Gabriel et al., 1997, Enders et al., 1997) so we used an approximate dose reduction factor of 20 (20 mg/kg) to 80 (5 mg/kg) in our experiments.
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2.5. Electrophysiological analysis
Nerve conduction tests were performed by a blinded investigator on the day before immunisation (-1) and on day 18 (maximum of natural disease course) p.i. The rats were anesthetized intraperitoneally (i.p.) with xylazine and ketamine (10 mg/kg and 50 mg/kg
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respectively). Using a fully digital recording Keypoint apparatus (Dantec, Skovlunde, Denmark) and paired needle electrodes inserted into the sciatic notch (hip; proximal) or the
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popliteal fossa (distal), the sciatic nerve was stimulated with supramaximal rectangular pulses of 0.05 ms duration and the resulting compound muscle action potential (cMAP) was
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recorded from needle electrodes placed subcutaneously over the dorsal foot muscles. A ground electrode was placed between the distal stimulating electrode and the active recording
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electrode. To calculate the motor nerve conduction velocity (mNCV), the distance between
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stimulating cathodes was divided by the difference of the latency. Similarly, the persistence and minimum latency of 10 F-waves evoked by stimulation at the popliteal fossa were
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recorded for the right side (Tuck et al., 1982, Taylor et al., 2003). Temperature differences were minimized by conducting the study as soon as the anesthesia had taken effect and by warming the leg with a heating lamp.
2.6. Histopathological assessment and immunohistochemistry After transcardial perfusion with PBS (Gibco) on day 18 or day 19 p.i. the two sciatic nerves were dissected and their segments were embedded in Tissue-Tek OCT Compound and snapfrozen in liquid nitrogen. For histopathology assessment, rat tissue was sectioned (8 µm) on a 8
ACCEPTED MANUSCRIPT cryostat (Leica Biosystems) and mounted on glass slides (Hartenstein, Würzburg, Germany). For the immunohistochemical staining, cryostat sections were fixated in acetone at 20°C for 10 minutes and were exposed to the mouse monoclonal antibodies (mAb) anti-rat 15-6A1 (Hycultec, Pan T-Cells CD3 1:100) and anti-rat ED1 (Hycultec, anti-CD68, macrophages, 1:100) using the avidin-biotin technique (Dako ARK KIT for Mouse Primary AB). The
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omission of the primary antibodies served as negative control. Specificity of the staining was also controlled on sections of peripheral lymphoid organs. The numbers of positive cells were counted at ×40 magnification for 12 sections per animal. The average results are expressed as cells per mm2 tissue section.
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For immunohistochemical stainings, cryostat sections were fixated in acetone at 20°C for 10 minutes and were exposed to the following monoclonal antibodies (mAb) anti-ICAM-1
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(1:100, Abcam, ab127160), anti-C3 (1:100, Abcam, ab11887), claudin-5 (1:100, Thermo Fisher Scientific, 4C3C2).
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Secondary antibodies conjugated with Alexa 555 (1:1000) or Alexa 488 (1:1000)
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(ThermoFisher, Schwerte, Germany) were used according to manufacturer’s protocol and DAPI (4',6' diamino-2-phenylindole·2HCl, Biozol, Eching, Germany) was used for
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fluorescent staining of DNA. Fluorescent signals were detected using an inverted fluorescence
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microscope (BX51; Olympus, Tokyo, Japan) equipped with an Olympus DP50 digital camera. For assessment of demyelination, lesions were identified by the accumulation of nuclei and absence of FluoroMyelinTM Red fluorescent stain (1:300, Molecular Probes) performed according to the manufacturer’s protocol. Images (20x-magnification) of twelve transverse sections of the sciatic nerve from each animal were digitally generated (Cell F 5.1, Olympus, Tokyo, Japan). The percentage of the area of demyelination per section was determined using image analysis software (ImageJ, National institutes of Health, Bethesda, USA). For histopathological assessment and immunohistochemistry, slides were blinded by a notinvolved third person and labelled with a numeric-code, which was unblinded after analysis. 9
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2.7. Isolation of mononuclear cells from lymph nodes and FACS analyses The inguinal lymph nodes were removed after transcardial perfusion with PBS (Gibco) on day 10 p.i. for the preventive setting under aseptic conditions. Single cell suspensions of mononuclear cells (MNC) from individual rats were prepared separately (preventive setting
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n=4/group). We evaluated the frequency of CD4+ T cells, CD11b+ cells, CD4+CD11b+ dendritic cells (DCs), CD4+ CD25+ FoxP3+ regulatory T-cells (Tregs) and CD4+CD11bMHCII+ plasmacytoid DCs by fluorescence-activated cell sorting (FACS) staining (eBioscience, San Diego, CA). FACS analyses were performed with a FACS Canto II (BD
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Pharmingen, Heidelberg, Germany) machine and FlowJo software (Tree Star). Monoclonal antibodies purchased from BD Pharmingen or eBioscience were used to detect CD4, CD11b,
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CD25 and MHC-II in accordance with the manufacturer´s instructions. Intracellular staining for Foxp3 was performed using the Foxp3 Staining Set (eBioscience) according to the
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manufacturer’s instructions.
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2.8 Tissue preparation, RNA isolation and gene expression analyses with quantitative rt-PCR
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Total RNA was isolated from the sciatic nerve samples of rats 10 days p.i. (n=8) using the RNeasy Mini extraction kit (Qiagen, Hilden, Germany). All samples were treated with the
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RNA Stabilization Reagent (RNAlater, Qiagen, Hilden, Germany) at 37 °C overnight and stored at −80 °C until use. Total RNA was reverse-transcribed into cDNA according to the manufacturer's protocol for the Reverse Transcription System (Promega, Madison, WI). IFNgamma, TNF-alpha, IL-10 and IL-4 mRNA expression levels were analyzed by quantitative RT-PCR according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). Sequence-specific primers and probes were designed using pre-developed TaqMan® assay reagents (Applied Biosystems). rt-PCR amplifications were carried out using the real-time PCR System 7500 (Applied Biosystems). In brief, real-time PCR for RNA-to-CTTM 1-Step 10
ACCEPTED MANUSCRIPT was performed according to the following amplification protocol: 25°C for 5 min, 42°C for 30 min and 82°C for 5min (for cDNA synthesis), 95°C for 3 min (transcriptase inactivation), followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. β-Actin was used to normalize relative mRNA expression. To compare each mRNA and housekeeping β-Actin mRNA expression, the relative expression of PCR products was determined using the ΔΔCt method
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(Schmittgen and Livak, 2008). Each experiment was performed in duplicate and the mean Ct was used in the equation.
2.9. Statistical methods
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Statistical analyses were performed by GraphPad Prism 6 software (GraphPad Software Inc., San Diego, USA). Data are provided as mean ± SEM (standard error of mean). Differences
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between pairs of groups were tested by Student’s t-test. Differences between three or more groups were tested by one-factor analysis of variance (ANOVA). Area under the curve (AUC)
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was calculated for clinical courses and analysed by one-way analysis of variance (ANOVA)
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combined with Tukey`s multiple comparison test. In all experiments, a P value of < 0.05 was
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significant.
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defined as statistically significant, and P < 0.0001 was considered as highly statistically
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ACCEPTED MANUSCRIPT 3. Results
3.1. Intrathecal human immunoglobulins applied in a therapeutic concept attenuate the clinical course of experimental autoimmune neuritis After immunisation with P2 protein peptide 53-78, clinical signs of EAN begun around day
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10 p.i. At days 11, 13 and 15 p. i. 0.9% NaCl or human immunoglobulins were applied intrathecally. The incidence of EAN for the control group was 100 % and the groups receiving human immunoglobulins showed an incidence of 100 % (5 mg/kg), 75 % (10 mg/kg) or 66 % (20 mg/kg) respectively. A dose-dependent amelioration of EAN clinical
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signs was documented (AUC, NaCl-treated vs. 20 mg/kg hIgs ***p<0.0001) (Figure 1). There was a non-significant reduction of body weight of sham-treated controls during the
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course of EAN as compared to rats treated with hIgs, in which body weight remained constant
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(data not shown).
3.2. Intrathecal human immunoglobulins improve mostly proximal but also distal nerve
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conduction
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To elucidate the effect of intrathecal immunoglobulins regarding proximal and distal demyelination, we performed electrophysiological measurements of the sciatic nerve at the
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maximum of the clinical course (day 18 p.i.) as described in material and methods. The following measures were evaluated: 1) spinal root involvement (proximal demyelination), depicted by F-wave persistence and F-wave latencies and 2) degree of distal demyelination, as implied by the reduction of the motor nerve conduction velocity (mNCV). Persistence of F-wave response, as defined by the number of F-waves elicited after 10 distal stimulations, was 40 % at the peak of disease (day 18 p.i.) for the NaCl-treated rats and 50 % for the 5 mg/kg intrathecal Igs-treated group (non-significant compared to control group). On the contrary, persistence of F-wave response was 95 % for the 10 mg/kg and 20 mg/kg 12
ACCEPTED MANUSCRIPT intrathecal Igs-treated groups (***p< 0.0001 compared to control group) consistent with early proximal sciatic nerve involvement in control animals and indicating the crucial protective role of immunoglobulins injected intrathecally against inflammatory root involvement (Figure 2A, B). The average minimum latencies of the elicitable F-waves were slightly prolonged in NaCl-
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treated group and in 5 mg/kg immunoglobulins-treated group between the peak of disease (day 18 p.i.) and day -1 p.i., whereas there was no difference for the 10 mg/kg hIg-treated group and the 20 mg/kg hIg-treated-group (data not shown).
mNCV was significantly reduced in the NaCl-treated rats (mean mNCV on day 18 p.i. 29, 7
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m/s vs. day -1 p.i. 50.4 m/s, ****p < 0.0001, n=4), whereas no difference of the mean mNCV on day -1 was seen for hIg-treated groups (Figure 2C), indicating the additional protective
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role of intrathecal application of immunoglobulin against distal demyelination.
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3.3. Intrathecal human immunoglobulin reduces histological signs of demyelination and T cell and macrophage infiltration in the sciatic nerve
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Next, we analyzed if the electrophysiological reduction of proximal and distal demyelination
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correlates with histological signs of myelination. Figure 3 shows representative pictures of FluoroMyelinTM staining. Administration of 10 and 20 mg/kg intrathecal immunoglobulins
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significantly reduced the percentage of demyelination of the sciatic nerves when compared to NaCl-treated control rats (***p<0.0001, n=4, Figure 3). Next, we showed that clinical improvement correlates with the reduction of inflammatory infiltration of the PNS. Histological data showing inflammation within the PNS are depicted in Figure 4. Intrathecal administration of 20 mg/kg immunoglobulins on days 11, 13 and 15 p.i. significantly reduced infiltration of macrophages and lymphocytes in the sciatic nerves compared to NaCl-treated control group, when examined on day 18 p.i. (***p<0.0001). Lower concentrations of immunoglobulins (5 and 10 mg/kg) showed a less pronounced 13
ACCEPTED MANUSCRIPT reduction of inflammatory cells (Figure 4). Interestingly, inflammatory infiltrates were reduced both in the distal and proximal parts of the sciatic nerves (data not shown).
3.4.
Intrathecal
immunoglobulins
preserve
ICAM-1-dependent
blood-nerve-barrier
permeability and reduce C3 expression in peripheral nerves
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Thereafter, we surveyed possible immunological mechanisms for the proximal but also distal reduction of infiltrating inflammatory cells in the peripheral nerves after intrathecal immunoglobulin treatment.
Among the multiple reported effects of immunoglobulins applied systemically are the
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reduction of endothelial transmigration molecules such as ICAM-1 (intercellular adhesion molecule). and of the C3 complement fragment (Basta et al., 1989).
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ICAM-1 expression was significantly reduced for 20 mg/kg-treated rats correlating with the most prominent reduction of inflammatory infiltrates for this concentration (***p<0.0001,
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Figure 5). In order to distinguish blood nerve preservation effects of Ig on the blood nerve barrier, which are unrelated to inflammatory infiltration we analysed histological claudin-5
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treatment (Figure 6).
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expression in the peripheral nerves, which showed no significant differences after Ig
Finally, we analysed C3 fragment expression in the sciatic nerves at the peak of disease, was
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significantly reduced after intrathecal immunoglobulin expression (***p<0.0001, Figure 7).
3.5. Preventive intrathecal immunoglobulin treatment reduced clinical, electrophysiological and histological EAN signs Therapeutic intervention with intrathecal immunoglobulins showed a remarkable effect in clinical, electrophysiological and histological signs of neuritis. The question arises if preventive intrathecal immunoglobulin injection at the EAN induction phase (days 0, 2, 4 p.i.) ameliorates clinical, electrophysiological and histological signs of dysimmune neuritis. As 14
ACCEPTED MANUSCRIPT shown in Figure 8 intrathecal immunoglobulins reduced EAN but their effect was more prominent for the low concentrations of 5 and 10 mg/kg (*p< 0.05). This effect was confirmed in electrophysiological and histological terms as only the lower concentrations of intrathecal immunoglobulins showed a preservation of mNCV on day 19 p.i. and a reduction
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of the inflammatory infiltrates (Figure 8 B, C, D).
3.6. Intrathecal immunoglobulins in a preventive concept reduce early C3 expression (day 10 p.i.) and induce an early Th2 cytokine shift in peripheral nerves
We showed above that immunoglobulins applied intrathecally in a therapeutic concept induce
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a dose-dependent reduction of C3 fragment expression and a preservation of blood-nervebarrier at disease maximum.
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In the preventive concept, the lowest concentration of hIg showed the most prominent effect implying possibly different mechanisms of action in this EAN phase. We proceeded to test
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the mechanisms of action of intrathecal immunoglobulins applied in the induction EAN phase by analyzing histological C3 fragment and ICAM expression as well as Th1 and Th2 cytokine
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profiles at day 10 p.i. (before clinical neuritis signs).
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At day 10 p.i. there was no statistically significant difference in the inflammatory infiltrates, ICAM or claudin-5 expression between NaCl-treated and hIg treated rats (data not shown).
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However, in the rats treated with a lower concentration of human immunoglobulins a statistically significant reduction of C3 histological expression in sciatic nerves was still induced just before disease symptoms started (Figure 9A). Furthermore, a Th2 cytokine shift was observed for the lower concentration of 5 mg/kg at that time point, as only IL-10 and IL4 mRNA expression showed a statistically significant increase before disease initiation (Figure 9 B, C, D, E). In order to detect possible peripheral effects of intrathecally applied immunoglobulins, we also isolated periherally mononuclear cells from the inguinal lymph nodes at day 10 p.i. and 15
ACCEPTED MANUSCRIPT analyzed them with FACS analysis. We evaluated the frequency of CD4+ T cells, CD11b+ cells, CD4+CD11b+ dendritic cells (DCs), CD4+ CD25+ FoxP3+ regulatory T-cells (Tregs) and CD4+CD11b-MHCII+ plasmacytoid DCs. No statistically significant differences were found
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between immunoglobulin and NaCl-treated rats for all populations tested (data not shown).
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ACCEPTED MANUSCRIPT 4. Discussion
In the current study, we present a new perspective of the classical treatment of autoimmune neuropathies with immunoglobulins using the alternative route of intrathecal application. This application route has not yet been used for immunoglobulins in autoimmune diseases of the
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peripheral nervous system although the first sites of inflammation and increase of blood-nerve barrier permeability are the proximal nerve roots, as indicated by the early intrathecal protein increase found in these patients (Hughes et al., 2006, Kerasnoudis et al., 2014). The intrathecal injection of a 98% total IgG protein preparation in low concentrations exerted
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unique immunomodulatory and anti-inflammatory actions in our model.
In the therapeutic concept, intrathecal immunoglobulins achieve a dose-dependent
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improvement of clinical signs, which correlates with a histological reduction of inflammatory infiltrates in the sciatic nerves.
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Firstly, we investigated whether this anti-inflammatory effect of intrathecal immunoglobulins is focused near the injection site (close to the in the nerve roots) or rather distally.
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Electrophysiologically, we found a prominent preservation of F-wave persistence as an
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indicator of an improvement of proximal demyelination as well as an unchanged motor conduction velocity as a sign of a distal preservation of myelination. These findings were
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histologically confirmed by large-field immunostaining for intact myelin, yet a thorough histological analysis using electron microscopy was not performed. We conclude that intrathecal injection in the effector phase reduces inflammatory activity beginning at the injections sites and extending into the whole nerve thereby achieving sufficient improvement of electrophysiological signs of demyelination. In order to dissect possible mechanisms of action of the local intrathecal injection of immunoglobulins, we analyzed possible immunomodulatory mechanisms in the peripheral nervous system reported before for systemically applied immunoglobulins. 17
ACCEPTED MANUSCRIPT Firstly, we showed a local reduction of C3 fragment expression in the peripheral nerves at the maximum of disease. This effect has been already described for systemically applied immunoglobulins and has been attributed to the F(ab')2 region of IgG. This region is shown to neutralize activated complement proteins, including C3a and inhibit the uptake of C3b and C4b onto the cell surface, thereby preventing complement-mediated tissue damage (Basta et
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al., 1989). However, for the intravenous application of human immunoglobulins, high concentrations are needed to exert these peripheral and local anti-inflammatory actions. In our model the disrupted blood-nerve-barrier during inflammation in combination with the local injection of immunoglobulins enables low concentrations of immunoglobulins to exert these
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anti-inflammatory actions.
Furthermore, the reduction of inflammatory infiltrates correlated with the downregulation in
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the histological expression of ICAM molecule in the peripheral nerves. This effect has been reported before for patients with autoimmune dermatomyositis in muscle biopsies after
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systemically immunoglobulin treatment (Rigal et al., 1994) as well as in sera of GBS patients after IVIg infusion (Sharief et al., 1999). Dermatomyositis studies revealed a downregulation
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of ICAM gene expression in treatment-responders was found (Raji et al., 2005). As described
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later in the discussion and based on our results this effect on ICAM-1 is secondary to the mononuclear cell infiltration.
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To our knowledge no reports exist on the effects of Ig on molecules preserving the bloodnerve barrier during inflammation such as claudin-5. However, there are reports which describe a decrease of claudin-5 in the sural nerve of CIDP patients (Kanda et al., 2004, Shimizu et al., 2014). In our model, there was no influence on the histological expression of claudin-5 after Ig treatment neither on the therapeutic concept nor at the preventive concept, therefore we conclude that the main effects of intrathecal Ig treatment in our case rather stem from immunomodulatory/anti-inflammatory effects than from a direct effect on blood-nerve barrier permeability. 18
ACCEPTED MANUSCRIPT Finally, further aspects of local (intrathecal) immunomodulatory action of immunoglobulins on cellular inflammatory receptors (FasL-Fas), autoantibodies or Fc-gamma-receptors could surely play a role in our model. This fact points out the great potential of this application route in inflammatory neuropathies and has to be investigated in the future. One aspect of immunoglobulin treatment, which is highly relevant for further clinical
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application, is the correlation of dose and time-point of application with the clinical symptoms of neuritis. These aspects are nowadays individually adjusted for each patient in order to achieve clinical stability. The fact that immunoglobulins have an anti-inflammatory action at clinical CIDP relapse or in the acute GBS phase is well known. Whether they also have an
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immunomodulatory effect on the events which contribute to chronic autoimmune inflammation, with immunoglobulins finally leading to prevention of further relapses is not
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known. This question was investigated in our model by applying immunoglobulins in a preventive concept combined with immunisation.
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We show that the effects of immunoglobulins in this phase are still present but surprisingly not in a dose-dependent manner. Low concentrations of immunoglobulins applied
of
clinical
and
electrophysiological
findings.
By
examining
the
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improvement
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intrathecally are sufficient to attenuate clinical EAN signs, which correlated with an
immunomodulatory effects in the peripheral nerves at day 10 p.i. we were able to dissect the
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primary effects of immunoglobulins on ICAM-1 and C3 fragment without the secondary effects through the reduction of inflammatory infiltrates. Based on our findings, we conclude that immunoglobulins exert a direct effect on C3 fragment expression but probably only a secondary effect on ICAM-1 expression. The shift to Th2 cytokines already at this early time point emphasizes the preventive potential of immunoglobulins through a possible direct effect on cellular inflammatory receptors in the induction phase. The reason for this non-dose dependent effect in this phase could be a receptor-saturation-mediated effect in the noninflamed PNS at this early time point. Our findings imply that intrathecal immunoglobulins 19
ACCEPTED MANUSCRIPT could play a useful preventive role in autoimmune relapses but most importantly in lower concentrations. Concluding, we present a new perspective in immunoglobulin treatment in peripheral neuropathies by applying intrathecal immunoglobulin in a preventive and therapeutic concept. A transfer of our application model in human subjects could have many pitfalls, which were
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not obvious in the animal model. Firstly, the aspect of toxicity due to the high viscosity of immunoglobulin preparation and the existence of L-proline has to be taken into account. This can be solved by modifying these parameters in the immunoglobulin preparation. Furthermore, allergic reactions, which are described after intravenous infusion could be more
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pronounced after intrathecal injection. However, we believe that in the light of our current
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treatment option for neuritis patients.
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observations intrathecal application of immunoglobulin preparations could be a promising
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Acknowledgments - Conflicts of Interest and Source of Funding
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This study was supported by a scientific grant from CSL Behring.
Kalliopi Pitarokoili has received travel grants from Biogen Idec and Bayer Pharmaceuticals and speakers honoraria from Biogen Idec, Novartis and Bayer Pharmaceuticals. Felix Kohle, Oluwaseun Fatoba and Xiomara Pedreituria report no disclosures. Jeremias Motte received travel grants from Biogen Idec. R. Gold has received consultation fees and speakers honoraria from Bayer Schering, Biogen Idec, Merck Serono, Novartis, Sanofi-Aventis and TEVA. He also acknowledges grant support from Bayer Schering, Biogen Idec, Merck-Serono, Sanofi-
20
ACCEPTED MANUSCRIPT Aventis and TEVA. Min-Suk Yoon received consulting, speaker honoraria and scientific grant from CSL Behring, speaker honoraria from Grifols.
Figure Legends: Figure 1: Representative clinical course of EAN rats (n=4). EAN was induced in Lewis rats
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by immunisation on day 0 with P2 peptide 53-78 plus CFA. Rats received human immunoglobulins (hIgs) intrathecally at doses of 5 mg/kg, 10 mg/kg and 20 mg/kg on days 11,13 and 15 post immunisation. Control rats received NaCl 0.9% only. The experiment was repeated three times, each time with n=4/group. Mean values and SEM are depicted.
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Statistical analysis and p-value calculation were performed calculating the area under the curve. Comparisson of AUC was done using one-way ANOVA combined with Tukey`s
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multiple comparisons test. NaCl treated vs 20 mg/kg ***p< 0.0001.
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Figure 2: Representative traces of F waves at disease maximum (day 18 p.i.) from NaCltreated rats (A) and 20 mg/kg intrathecal Igs-treated rats (B). NaCl treated rats presented with
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a reduced persistence of F-wave response whereas Ig-treated rats showed no a normal
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response indicating the effect of effect of Igs in reducing proximal demyelination. C. Motor nerve conduction velocity of the sciatic nerve one day before immunisation (d-1) and at the
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peak of disease (day 18 p.i.) (n=4/group): After proximal and distal stimulation of the sciatic nerve the conduction velocity was calculated. A statistical significant reduction of the MNCV appeared only for the NaCl-treated group (n=4) but no difference in the mNCV was seen for all immunoglobulin-treated groups, indicating a protective role of immunoglobulins against distal demyelination. Mean values and SEM are depicted, p-values ****p< 0.0001. The experiments were repeated three times with n=4/group.
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ACCEPTED MANUSCRIPT Figure 3: Representative pictures and statistical analysis of Fluoromyelin staining for sciatic nerve transverse sections of rats (n=4/group) treated with immunoglobulins 5, 10 and 20 mg/kg and NaCl-treated animals, showing massive demyelination for NaCl-treated rats and preservation of myelin for Igs treated groups (***p<0.0001). The experiment was repeated
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three times with similar results. Scale bars indicate 100µm.
Figure 4: Mean numbers of A. CD68+ macrophages and B. T cells per mm2 sciatic nerve sections as measured by immunohistochemistry on day 18 p.i. from EAN rats (n=4/group)
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receiving intrathecal immunoglobulins in a therapeutic concept at different doses (5 mg/kg, 10 mg/kg, 20 mg/kg) and NaCl-treated rats at days 11, 13, 15 post immunisation. Mean
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three times with similar results.
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values and SEM are depicted, p-values***p<0.0001 *p<0.05. The experiment was repeated
Figure 5: Representative pictures and statistical analysis of ICAM staining for sciatic nerve
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transverse sections of rats (n=4/group) treated with immunoglobulins 5, 10 and 20 mg/kg and
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NaCl-treated animals, showing a reduction of ICAM histological expression for 10 and 20 mg/kg Ig-treated rats, which implies a protective secondary effect on blood-nerve-barrier
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permeability (***p<0.0001). The experiment was repeated three times with similar results. Scale bars indicate 100µm.
Figure 6: Representative pictures and statistical analysis of claudin-5 staining for sciatic nerve transverse sections of rats (n=4/group) treated with immunoglobulins 5, 10 and 20 mg/kg and NaCl-treated animals, showing a tendency towards no statistical significant difference in claudin-5 histological expression for Ig-treated rats. The experiment was repeated three times with similar results. Scale bars indicate 100µm. 22
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Figure 7: Representative pictures and statistical analysis of C3 staining for sciatic nerve transverse sections of rats (n=4/group) treated with immunoglobulins 5, 10 and 20 mg/kg and NaCl-treated animals, showing a reduction of C3 histological expression for 10 and 20 mg/kg Ig-treated rats (***p<0.0001). The experiment was repeated twice with similar results. Scale
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bars indicate 100µm.
Figure 8: A. Clinical course of EAN in Lewis rats after intrathecal immunoglobulins in a preventive concept. EAN was induced in Lewis rats by immunisation on day 0 with P2
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peptide 53-78 plus CFA. Rats received human immunoglobulins (hIgs) intrathecally at doses of 5 mg/kg, 10 mg/kg and 20 mg/kg on days 0, 2 and 4 post immunisation. Control rats
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received NaCl 0.9% only. Pooled results from three independent experiments (n=4-12/group). Mean values and SEM are depicted. AUC, NaCl treated vs 5 mg/kg *p< 0.05.
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B. Motor nerve conduction velocity of the sciatic nerve one day before immunisation (d-1) and at day 19 p.i. (n=4/group): After proximal and distal stimulation of the sciatic nerve the
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conduction velocity was calculated. A statistical significant reduction of the MNCV appeared
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only for the NaCl-treated group (n=4) but no difference in the mNCV was seen for all immunoglobulin-treated groups, indicating a protective role of immunoglobulins against
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distal demyelination. Mean values and SEM are depicted, p-values ***p< 0.0001. Pooled results from three independent experiments (n=4-12/group). C, D Mean numbers of C. CD68+ macrophages and D. T cells per mm2 sciatic nerve sections as measured by immunohistochemistry on day 19 p.i. from EAN rats (n=8/group) receiving intrathecal immunoglobulins in a therapeutic concept at different doses (5, 10 and 20 mg/kg) and NaCl-treated rats at days 0, 2, 4 post immunisation. Mean values and SEM are depicted, p-values****p<0.0001.
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ACCEPTED MANUSCRIPT Figure 9 A. C3 histological expression at day 10 p.i. for sciatic nerve transverse sections of rats (n=4/group) treated with immunoglobulins 5 and 40 mg/kg and NaCl-treated animals, showing a reduction of C3 histological expression only for 5 mg/kg hIg-treated rats (***p<0.0001, **p<0.001). Relative gene expression of Th1 cytokines (B. IFN-gamma,D. TNF-alpha) and Th2 cytokines
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(C. IL10, D. IL-4) in sciatic nerves at day 10 p.i. after application of 5 and 40 mg/kg hIgs showing a statitstical signifcant increase of IL-10 and IL-4 cytokines as a sign of a Th2
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ACCEPTED MANUSCRIPT Highlights Intrathecal immunoglobulins (Ig) access the main site of inflammation in EAN Intrathecal Ig ameliorates clinical signs in Lewis rats neuritis model It reduces autoimmune inflammation, demyelination and complement activation In a preventive concent at low concentrations it induces a Th2 cytokine shift
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Intrathecal Ig could be a novel treatment option for autoimmune neuropathies
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