Oxygen radicals and severity of GBS 1

OXYGEN RADICAL PRODUCTION AND SEVERITY OF THE GUILLAIN-

BARRÉ SYNDROME

Natalia Mossberga, Oluf Andersena, Staffan Nilssonb, Claes Dahlgrenc, Kristoffer

Hellstrandd, Magnus Lindhd, Åke Svedhemd, Tomas Bergströmd and Charlotta Movitzd

aDepartment of Neuroscience and Physiology, Sahlgrenska University Hospital, Göteborg,

Sweden; bDepartment of Mathematical Statistics, Chalmers University of Technology,

Göteborg, Sweden; cDepartment of Rheumatology and Inflammation Research, Göteborg

University, Sweden; dDepartment of Infectious Medicine, Göteborg university, Sweden

Correspondence to Oluf Andersen, Neurology-Sahlgrenska, Gröna Stråket 11 3tr, Sahlgrenska

University Hospital, 413 45 Göteborg, Sweden. Oxygen radicals and severity of GBS 2

ABSTRACT

The NADPH oxidase-dependent formation of reactive oxygen species (“oxygen radicals”) by phagocytic cells constitutes an important part of the innate immune defence against microorganisms. Recent studies in animal models imply that a deficient function of the

NADPH oxidase may be linked to the development of autoimmunity, but a link between oxygen radical production and severity of autoimmune disease in humans has not been established. We have examined the oxygen radical production in peripheral blood leukocytes from patients with the Guillain-Barré syndrome (GBS). Leukocytes from GBS patients in a stationary phase 1-5 years after their acute episode were activated by the formyl peptide receptor (FPR) ligand formyl-Met-Leu-Phe (fMLF) or the closely related formyl peptide like receptor 1 (FPRL1) ligand Trp-Lys-Tyr-Met-Val-Met-NH2 (WKYMVM). The patients were dichotomized according to severity by 1) the requirement of intensive care unit treatment and,

2) the ability to walk independently after 3 months . Our data show that the amount of superoxide release following challenge with either of the two agonists fMLF and WKYMVM was significantly lower in patients requiring intensive care unit treatment or unable to walk after 3 months . Results obtained with the global activator phorbol myristate acetate, as well as with fMLF in TNF -primed leukocytes, suggested that the deficiency of oxygen radical production in patients with severe GBS was the result of a specific deficiency of radical production in response to FPR/FPRL1 ligands rather than an inherent deficiency of NADPH oxidase function. Oxygen radicals and severity of GBS 3

Key words: Guillain-Barré syndrome; autoimmunity; NADPH oxidase; reactive oxygen species; phagocytes; severity Oxygen radicals and severity of GBS 4

Introduction

The Guillain-Barré syndrome (GBS) is a disease of the peripheral nervous system that is characterized by segmental demyelination and infiltration of mononuclear cells in peripheral nerves and nerve roots with axonal degeneration in severe lesions (Asbury et al., 1969). GBS is often preceded by an infectious illness. Campylobacter jejuni is the most commonly identified infectious trigger for GBS, followed by viral agents like cytomegalovirus (CMV) or

Epstein-Barr virus (EBV) (Hadden et al., 2001). Molecular mimicry between microbial antigens and host nervous tissue appears to be important in the pathophysiology of GBS, which is thus considered an organ-specific autoimmune disease (Ang et al., 2004).

Reactive oxygen species (“oxygen radicals”) are produced by phagocytic cells and form a pivotal part of the innate immune defence against microorganisms, primarily bacteria but also certain viruses (Babior, 2000) (Klebanoff and Coombs, 1992). Recent studies in animal models imply that a deficiency of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which initiates oxygen radical production in phagocytes, may be related to the development of autoimmunity. A polymorphism of Ncf1, which encodes a component of the

NADPH oxidase (p47phox), is thus associated with impaired oxygen radical production along with severe autoimmune arthritis and encephalitis in rodents (Olofsson et al., 2003) (Hultqvist et al., 2004).

The putative link between the NADPH oxidase-dependent oxygen radical formation and the severity of autoimmunity incited us to investigate the capacity of leukocytes from patients with a history of GBS to produce radicals in response to NADPH oxidase-inducing agents in vitro. Our findings imply that a constitutively deficient oxygen radical induction is associated with the severity of GBS. Oxygen radicals and severity of GBS 5

Materials and methods

Patients

The patients (n=23,16 males and 7 females) had been treated for their acute episode of GBS at

Sahlgrenska University Hospital, Göteborg 1-5 years before this study. All patients fulfilled the Asbury criteria for GBS (Asbury and Cornblath, 1990). Healthy control subjects were the patients’ spouses (n=17) or age-matched healthy subjects (n=6) with mean age 51±17 years.

The study was approved by the Medical Ethics Committee and written informed consent was obtained from all participators.

Subdivision according to severity in the acute phase was performed by two criteria: the ability to walk independently after 3 months, or treatment in an intensive care unit (ICU) (yes/no).

Subdivision according to severity of residual symptoms at re-examination was based on a modified Medical Research Council (MRC) scale (Kleyweg et al., 1991) (sum score >2 or ≤

2, table 1). Subdivision according to preceding infection was based on serology (table 1). A diagnosis of Campylobacter jejuni infection was based on a positive IgM titer or a significant rise of IgG titers. Campylobacter antibodies were detected by DIG-ELISA utilising an outer membrane protein of glycoprotein type (Svedhem et al., 1983) Series of sera over time showing production of IgM antibodies, or increased titers of Campylobacter IgG antibodies were used to determine the presence of an actual or recent infection. A diagnosis of viral infections was based on significant IgM titers in serum.

Peptides and reagents Oxygen radicals and severity of GBS 6

Trp-Lys-Tyr-Met-Val-Met-NH2 (WKYMVM) was from Alta Bioscience (University of

Birmingham, United Kingdom). N-formyl-Met-Leu-Phe (fMLF) and phorbol myristate acetate (PMA) were from Sigma Chemical Co. (St. Louis, Missouri). Tumor necrosis factor alpha (TNF-) was from Boehringer-Mannheim (Mannheim, Germany).

Isolation of leukocytes

Samples of heparinized venous blood were collected and coded for the blind analysis.

Peripheral leukocytes (neutrophils, monocytes and lymphocytes) were isolated by dextran sedimentation and hypotonic lysis of remaining erythrocytes as described elsewhere (Boyum,

1968) (Metcalf et al., 1986). Leukocytes were resuspended (107 cells/ml) in Krebs-Ringer buffer (Dahlgren and Karlsson, 1999). No significant differences in leukocyte distribution were detected between patients and controls or between the clinical subgroups of patients

(data not shown).

NADPH oxidase activity of leukocytes

Leukocyte production of superoxide anion, which is the initial oxygen radical formed by the

NADPH oxidase (Dahlgren and Karlsson, 1999), was assessed by isoluminol/luminol- enhanced chemiluminescence (CL) technique (Lundqvist and Dahlgren, 1996). The CL was measured in a Biolumat LB 9505 (Berthold Co.,Wildbad, Germany), using a 1 ml reaction mixture containing 106 leukocytes, horseradish peroxidase (4 U/ml) and isoluminol or luminol

(20 µM). The cells were activated by the addition of 10-7 M fMLF, 10-7 M WKYMVM or

5x10-8 M PMA or primed by incubation with 10 ng/ml TNF- for 20 minutes at 37°C before stimulation with 10-7 M fMLF, as described in detail elsewhere (Bylund et al., 2004). Oxygen radicals and severity of GBS 7

Results

Production of superoxide anions in clinical subgroups of GBS

We compared the superoxide anion production of leukocytes isolated from GBS patients with different severity of disease. The patients were dichotomized according to 1) able to walk independently 3 months after onset, 2) requirement of ICU with respirator facilities , 3) mobility score according to a modified MRC scale at follow-up, and 4) Campylobacter jejuni serology. Patients who had required ICU treatment during the acute phase of GBS produced significantly lower amounts of oxygen radicals after stimulation with the NADPH oxidase- triggering peptides fMLF and WKYMVM (Figure 1A and table 2), compared with patients who had not required ICU (p = 0.009). The other two severity measures, the ability to walk after 3 months (Figure 1B) and MRC scale (data not shown), were in strong agreement with the ICU parameter, and also revealed a significant correlation between diminished oxygen radical production and severity of GBS. No difference in superoxide anion production between these subgroups of GBS patients was detected after stimulation with PMA (Table 2), and no significant differences in superoxide anion production were observed between the total group of GBS patients (n=23) and their respective controls for any of the activators used

(p>0.2, data not shown).

The influence of age, gender, and Campylobacter jejuni serological status was evaluated using logistic regression analysis with either the fMLF- or WKYMVM-induced oxygen radical production as independent variables and severity of disease (ICU yes/no) as a Oxygen radicals and severity of GBS 8 dependent variable. No difference.in oxygen radical production was observed between patients with and without a preceding Campylobacter jejuni infection (p=0.053 for

WKYMVM-stimulated cells, data not shown). Only fMLF- (p=0.03) and WKYMVM-

(p=0.03) induced oxygen radical production remained significant predictors of disease severity in the multivariate analysis.

Production of oxygen radicals after TNF- induced priming

These findings suggested that leukocytes from clinical subgroups of patients with GBS differ in their capacity to generate oxygen radicals in response to fMLF and WKYMVM, which are ligands for formyl peptide receptors (FPR) (Berkow et al., 1987). Cells primed by pretreatment with TNF-α mobilize stored surface receptors of fMLF (FPR), which optimizes cellular responsiveness to this bacterial product (Bylund et al., 2004). To clarify whether the diminished oxygen radical production was restored by mobilization of FPR receptors, we treated leukocytes with TNF- prior to exposure to fMLF. As shown in Figure 1C and D, this priming procedure efficiently corrected the deficient oxygen radical production in patients with severe GBS. Oxygen radicals and severity of GBS 9

Discussion

We studied oxygen radical production in response to NADPH oxidase-activating agents in leukocytes from patients in the convalescence state after GBS. The results imply that patients who had a severe GBS show a constitutionally lower oxygen radical production. This observation is in analogy with the finding that a polymorphism in Ncf1, which encodes one of the cytosolic components of the NADPH oxidase, is associated with severe experimental allergic encephalomyelitis (EAE) and collagen-induced arthritis in rodents along with a reduced NADPH oxidase function (Olofsson et al., 2003) (Hultqvist et al., 2004).

A difference in radical production between clinical subgroups of patients with GBS was only detected following stimulation with fMLF and WKYMVM. These peptides are ligands for transmembrane G-protein coupled receptors that belong to the FPR family: fMLF is an N- terminal formylated tripeptide which activates the NADPH oxidase by interaction with FPR, whereas the hexapeptide WKYMVM binds both to receptors of the FPR-like 1 (FPRL1) and

FPR-like 2 (FPRL2) subtypes (Fu et al., 2006). In contrast, the severity of GBS was not influenced by the oxygen radical production of leukocytes treated with PMA, a membrane- permeable PKC activator (Wolfson et al., 1985). PKC is an intracellular transduction kinase, which is located downstream of FPR and FPRL receptors and upstream of the NADPH oxidase, and thus activates the oxidase independently of FPR/FPRL; these findings therefore suggest that the overall function of the oxidase is intact in patients with severe GBS. The results obtained using PMA-activated leukocytes also imply that the reduced responsiveness to NADPH oxidase inducers in patients with severe GBS is located upstream of PKC. Oxygen radicals and severity of GBS 10

A tentative conclusion from our study is that leukocytes from patients severe GBS are functionally impaired regarding FPR-mediated activation of the NADPH oxidase. After exposure to TNF- leukocytes become primed with respect to NADPH oxidase activity upon subsequent stimulation (Berkow et al., 1987), which reportedly is the result of mobilization of intracellular FPR to the plasma membrane (Bylund et al., 2004) (Bylund et al., 2002). The finding that the priming procedure efficiently restored NADPH oxidase function in patients with severe GBS thus underlines that the observed deficiency of oxygen radical production may be related to a deficient expression or function of these receptors. It should be pointed out, however, that our results do not exclude the involvement of other mechanisms, and that the NADPH oxidase dysfunction in GBS should be the subject of further study.

Superoxide anion is the initial oxygen radical formed by the NADPH oxidase, and this anion is the substrate for a series of reactive oxygen species. Superoxide anion is converted to hydrogen peroxide, which is the substrate for halide compounds in reactions catalyzed by myeloperoxidase (MPO) (Klebanoff, 1999). Myeloperoxidase (MPO) is a granule-localized protein found both in neutrophils and monocytes. These granules also contain microbicidal substances that are delivered into the phagosome by membrane fusion. In the presence of

MPO, hydrogen peroxide can oxidize halides forming hypochloric acid. Hypochloric acid mediates the oxidation and halogenation of cellular constituents but is also of importance during down-regulation of auto-immune reactions. By reacting with taurine, an amino acid present in most mammalian tissues, taurine chloramines are synthesized. Taurine chloramines have been suggested to be of importance during elimination of auto-immune T-cells clones by inhibiting lymphocyte proliferation and cytokine production (Park et al., 2002). Recently, low levels of MPO activity were reported in leukocytes from patients with multiple sclerosis

(Ramsaransing et al., 2003). In the present study, no differences in the MPO activity of Oxygen radicals and severity of GBS 11 leukocytes, measured as the MPO- and hydrogen peroxide-dependent oxidation of 4- aminantipyrine (Metcalf et al., 1986), were observed between clinical subgroups of GBS (data not shown), thus suggesting that the severity of GBS is not determined by MPO activity.

The severity of GBS is influenced by some of the preceding infections, with a delayed early recovery reported after CMV associated GBS, and a higher risk of residual symptoms after campylobacter associated GBS (Visser et al., 1999). On the other hand, few host factors were reported to influence the outcome of GBS. Recently, its severity was reported to be associated with certain haplotypes related to high activity of the mannose-binding lectin (Geleijns et al.,

2006). In the present study, a tendency towards a more severe course with preceding campylobacter infection was not confirmed in a multifactorial analysis, where only the oxygen radical production following stimulation with fMLF and WKYMVM remained significantly associated with the GBS severity variables.

It is well-known that ROS contributes to the tissue damage in several diseases (Babior, 2000)

(Minohara et al., 2006). However, ambiguity between bactericidal and injurious effects of

ROS was reported in other systems, including the effects of NO (Andrews et al., 1999). As a possíble explanation of our results, a defect in oxygen radical production has been suggested to enhance T-cell dependent autoimmunity (Hultqvist et al., 2007) (Gelderman et al., 2006).

Deficiency of the innate immune defence has been implicated in the pathogenesis of some autoimmune diseases (Etzioni, 2003), such as Crohn’s disease (van Heel et al., 2005). Thus, the innate system can be protective by clearing the pathogens that trigger or exacerbate the disease, or by regulating presentation of antigens to T lymphocytes, and defective eradication of microorganisms with a persisting antigen stimulation or failure in clearance of self-reactive lymphocytes can result in organ-specific autoimmune diseases (Carroll, 2001). Oxygen radicals and severity of GBS 12

In conclusion, our findings suggest that the individual capacity to generate oxygen radicals in response to FPR/FPRL ligands may determine the severity of GBS. As the design of the present study favoured detection of a constitutive factor rather than a disease-induced characteristic, this phenotype is probably genetically determined. As corroborated by the results of an experimental trial (Hultqvist et al., 2006), our findings may have implications not only for the understanding the pathogenesis of GBS and related autoimmune disorders, but may also form a basis for prophylactic or therapeutic intervention. Oxygen radicals and severity of GBS 13

Acknowledgements

This work was supported by grants from the Research Foundation of The Gothenburg

Multiple Sclerosis Society, The Swedish Medical Research Council, The Inga-Britt and Arne

Lundberg Research Foundation, and the Anna-Lisa and Bror Björnsson Foundation,

Göteborg, Sweden. Oxygen radicals and severity of GBS 14

References

Andrews, D.M., Matthews, V.B., Sammels, L.M., Carrello, A.C., McMinn, P.C., 1999. The severity of Murray Valley encephalitis in mice is linked to neutrophil infiltration and inducible nitric oxide synthase activity in the central nervous system. J Virol 73, 8781- 8790. Ang, C.W., Jacobs, B.C., Laman, J.D., 2004. The Guillain-Barre syndrome: a true case of molecular mimicry. Trends Immunol 25, 61-66. Asbury, A.K., Arnason, B.G., Adams, R.D., 1969. The inflammatory lesion in idiopathic polyneuritis. Its role in pathogenesis. Medicine (Baltimore) 48, 173-215. Asbury, A.K., Cornblath, D.R., 1990. Assessment of current diagnostic criteria for Guillain- Barre syndrome. Ann Neurol 27 Suppl, S21-24. Babior, B.M., 2000. Phagocytes and oxidative stress. Am J Med 109, 33-44. Berkow, R.L., Wang, D., Larrick, J.W., Dodson, R.W., Howard, T.H., 1987. Enhancement of neutrophil superoxide production by preincubation with recombinant human tumor necrosis factor. J Immunol 139, 3783-3791. Boyum, A., 1968. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl 97, 77-89. Bylund, J., Karlsson, A., Boulay, F., Dahlgren, C., 2002. Lipopolysaccharide-induced granule mobilization and priming of the neutrophil response to Helicobacter pylori peptide Hp(2-20), which activates formyl peptide receptor-like 1. Infect Immun 70, 2908- 2914. Bylund, J., Pellme, S., Fu, H., Mellqvist, U.H., Hellstrand, K., Karlsson, A., Dahlgren, C., 2004. Cytochalasin B triggers a novel pertussis toxin sensitive pathway in TNF-alpha primed neutrophils. BMC Cell Biol 5, 21. Carroll, M., 2001. Innate immunity in the etiopathology of autoimmunity. Nat Immunol 2, 1089-1090. Dahlgren, C., Karlsson, A., 1999. Respiratory burst in human neutrophils. J Immunol Methods 232, 3-14. Etzioni, A., 2003. Immune deficiency and autoimmunity. Autoimmun Rev 2, 364-369. Fu, H., Karlsson, J., Bylund, J., Movitz, C., Karlsson, A., Dahlgren, C., 2006. Ligand recognition and activation of formyl peptide receptors in neutrophils. J Leukoc Biol 79, 247-256. Gelderman, K.A., Hultqvist, M., Holmberg, J., Olofsson, P., Holmdahl, R., 2006. T cell surface redox levels determine T cell reactivity and arthritis susceptibility. Proc Natl Acad Sci U S A 103, 12831-12836. Geleijns, K., Roos, A., Houwing-Duistermaat, J.J., van Rijs, W., Tio-Gillen, A.P., Laman, J.D., van Doorn, P.A., Jacobs, B.C., 2006. Mannose-binding lectin contributes to the severity of Guillain-Barre syndrome. J Immunol 177, 4211-4217. Hadden, R.D., Karch, H., Hartung, H.P., Zielasek, J., Weissbrich, B., Schubert, J., Weishaupt, A., Cornblath, D.R., Swan, A.V., Hughes, R.A., Toyka, K.V., 2001. Preceding infections, immune factors, and outcome in Guillain-Barre syndrome. Neurology 56, 758-765. Hultqvist, M., Backlund, J., Bauer, K., Gelderman, K.A., Holmdahl, R., 2007. Lack of reactive oxygen species breaks T cell tolerance to collagen type II and allows development of arthritis in mice. J Immunol 179, 1431-1437. Hultqvist, M., Olofsson, P., Gelderman, K.A., Holmberg, J., Holmdahl, R., 2006. A new arthritis therapy with oxidative burst inducers. PLoS Med 3, e348. Oxygen radicals and severity of GBS 15

Hultqvist, M., Olofsson, P., Holmberg, J., Backstrom, B.T., Tordsson, J., Holmdahl, R., 2004. Enhanced autoimmunity, arthritis, and encephalomyelitis in mice with a reduced oxidative burst due to a mutation in the Ncf1 gene. Proc Natl Acad Sci U S A 101, 12646-12651. Klebanoff, S.J., 1999. Myeloperoxidase. Proc Assoc Am Physicians 111, 383-389. Klebanoff, S.J., Coombs, R.W., 1992. Viricidal effect of polymorphonuclear leukocytes on human immunodeficiency virus-1. Role of the myeloperoxidase system. J Clin Invest 89, 2014-2017. Kleyweg, R.P., van der Meche, F.G., Schmitz, P.I., 1991. Interobserver agreement in the assessment of muscle strength and functional abilities in Guillain-Barre syndrome. Muscle Nerve 14, 1103-1109. Lundqvist, H., Dahlgren, C., 1996. Isoluminol-enhanced chemiluminescence: a sensitive method to study the release of superoxide anion from human neutrophils. Free Radic Biol Med 20, 785-792. Metcalf, J.A., Gallin, J.I., Nauseef, W.M., K.Root, R., 1986. Laboratory Manual of Neutrophil Function. Minohara, M., Matsuoka, T., Li, W., Osoegawa, M., Ishizu, T., Ohyagi, Y., Kira, J., 2006. Upregulation of myeloperoxidase in patients with opticospinal multiple sclerosis: positive correlation with disease severity. J Neuroimmunol 178, 156-160. Olofsson, P., Holmberg, J., Tordsson, J., Lu, S., Akerstrom, B., Holmdahl, R., 2003. Positional identification of Ncf1 as a gene that regulates arthritis severity in rats. Nat Genet 33, 25-32. Park, E., Jia, J., Quinn, M.R., Schuller-Levis, G., 2002. Taurine chloramine inhibits lymphocyte proliferation and decreases cytokine production in activated human leukocytes. Clin Immunol 102, 179-184. Ramsaransing, G., Teelken, A., Prokopenko, V.M., Arutjunyan, A.V., De Keyser, J., 2003. Low leucocyte myeloperoxidase activity in patients with multiple sclerosis. J Neurol Neurosurg Psychiatry 74, 953-955. Svedhem, A., Gunnarsson, H., Kaijser, B., 1983. Diffusion-in-gel enzyme-linked immunosorbent assay for routine detection of IgG and IgM antibodies to Campylobacter jejuni. J Infect Dis 148, 82-92. van Heel, D.A., Ghosh, S., Hunt, K.A., Mathew, C.G., Forbes, A., Jewell, D.P., Playford, R.J., 2005. Synergy between TLR9 and NOD2 innate immune responses is lost in genetic Crohn's disease. Gut 54, 1553-1557. Visser, L.H., Schmitz, P.I., Meulstee, J., van Doorn, P.A., van der Meche, F.G., 1999. Prognostic factors of Guillain-Barre syndrome after intravenous immunoglobulin or plasma exchange. Dutch Guillain-Barre Study Group. Neurology 53, 598-604. Wolfson, M., McPhail, L.C., Nasrallah, V.N., Snyderman, R., 1985. Phorbol myristate acetate mediates redistribution of protein kinase C in human neutrophils: potential role in the activation of the respiratory burst enzyme. J Immunol 135, 2057-2062.

Oxygen radicals and severity of GBS 16

Table 1. GBS patient characteristics1

Sex (male/female) 16/7 Age, range/mean  SD (years) 23-81/53  17 Clinical variant: classical 19 Miller Fisher variant 4 EMG variant: axonal* 5 demyelinating 10 demyelinating with secondary axonal 5 normal 1 not performed 2 Severity: severe (TTIW*** ≥ 3 months) 9 not severe (TTIW < 3 months) 14 Required ICU yes 8 no 15 MRC score > 2 8 ≤ 2 15 Ganglioside antibodies 9 (4 n.a.) Infection serology: Campylobacter jejuni ** 16 EBV 3 CMV 2 mumps 2 RSV 1

Treatment: IVIG 8 plasmapheresis 6 IVIG + plasmapheresis 6 Untreated 3

1Demographics and modified MRC score refer to the time of re-examination, while clinical data apply to the acute GBS phase (Kleyweg et al., 1991). The following muscle groups were assessed: extension of arm; abduction/extension of the fingers; flexion of the hip; dorsal extension of the foot. 0 = normal, 1 = weak when tested against resistance (or with elevation and supination of arms), 2 = range of motion decreased with fatigue, 3 = unable to move Oxygen radicals and severity of GBS 17 complete range against gravity, 4 = paralysis of weaker synergy (flexion in legs), 5 = paralysis. Facial palsy, loss of position sense, sensory ataxia each =1. Loss of biceps or knee reflex each = 1. The scores are given for each half of the body. Dysarthria/dysphagia = 1.

Maximum score was 51.

*One patient had features of AMAN, i.e. pure motor neuropathology and anti-GM1 antibodies.

**Seven patients had positive serology for both Campylobacter and virus infections and one for both

Campylobacter and Borrelia.

***TTIW (time to independent walking) ≥ 3 months: able to walk independently after 3 months;

TTIW < 3 months: unable to walk independently after 3 months. Oxygen radicals and severity of GBS 18

Table 2. Superoxide anion production in leukocytes isolated from GBS patients subjected to intensive care unit treatment.1

- O2 production (Mcpm) Stimuli no ICU (n=15) ICU (n=8) p value fMLF 197  18.1 115 19.2 0.009** WKYMVM 170  17.6 96  12.1 0.009** PMA 621  22.0 617  34.8 0.9

1Leukocytes isolated from GBS patients were triggered with 10-7 M fMLF, 10-7 M

WKYMVM or 5x10-8 M PMA. The release of superoxide anions was measured by isoluminol/luminol-enhanced chemiluminescence. Data are presented as mean peak value ±

SEM. The statistical significance of difference in superoxide anion production between GBS patients with or without intensive care unit (ICU) treatment was determined using Student’s two-sample t-test. Oxygen radicals and severity of GBS 19

Legend to figure

Figure 1. Production of superoxide anion in clinical subgroups of GBS

Leukocytes isolated from clinical subgroups of GBS were stimulated by 10-7 M fMLF (A; patients with or without ICU treatment, B; patients with fast TTIW: able to walk independently after 3 month or slow TTIW: unable to walk independently after 3 months) or pre-incubated in presence of 10 ng/ml TNF- before stimulation with fMLF (C and D; patients with or without

ICU treatment). The production of superoxide anion was measured by isoluminol-amplified chemiluminescence. Responses are given as Mcpm (106 counts per minute) and show the average response ± SEM at different time points (A, B, C) or the peak values (D).

The statistical significance was determined using Student’s two-sample t-test.