A b n o rmally Decreased HbA1 c Can Be

Anti-thyroperoxidase antibodies from patients with Hashimoto's

encephalopathy bind to cerebellar astrocytes

Stéphanie Blanchin a,?,Christine Coffin b ,Fausto Viader c ,Jean Ruf d ,Pierre Carayon d ,

Francette Potier a ,Estelle Portier a ,Elisabeth Comby a ,Stéphane Allouche e ,

Yann Ollivier f ,Yves Reznik b ,Jean Jacques Ballet a

Laboratoire d ’Immunologie et d ’Immunopathologie,UPRES-EA 2128,CHU Clémenceau,14033Caen cedex,France

Service d ’Endocrinologie et Maladies Métaboliques,CHU C?te de Nacre,14033Caen cedex,France

Service de Neurologie,CHU C?te de Nacre,14033Caen cedex,France

INSERM U555,Facultéde médecine Timone,Universitéde la Méditerranée,13385,Marseille cedex 5,France

Laboratoire de Biochimie,CHU C?te de Nacre,14033Caen cedex,France f

Service de Médecine Interne,CHU C?te de Nacre,14033Caen cedex,France

Received 28June 2007;received in revised form 31July 2007;accepted 6August 2007

Abstract

A cohort of 10Hashimoto's encephalopathy (HE)patients,33patients with unrelated neurological symptoms,12Hashimoto's thyroiditis patients and 4healthy adult donors was studied to explore the neurological targets of anti-thyroperoxidase (TPO)autoantibodies (aAb)in HE.High levels of anti-TPO aAb were only detected in HE group's cerebrospinal fluids.In immunofluorescence assays on monkey brain cerebellum sections,both HE patients'sera and anti-TPO monoclonal antibodies (mAb)were able to bind cerebellar cells expressing glial fibrillary acid protein.Normal human astrocytes from primary cultures also reacted with anti-TPO mAb.Specific astrocyte binding of anti-TPO aAb suggests a role of these aAb in the HE pathogenesis.?2007Elsevier B.V .All rights reserved.

Keywords:Anti-thyroperoxidase antibodies;Astrocytes;Cerebrospinal fluid;Hashimoto's encephalopathy;Hashimoto's thyroiditis

1.Introduction

Hashimoto's encephalopathy (HE)is a rare,often misdiag-nosed and poorly understood corticosteroid-responsive neuro-logical syndrome occurring in patients with autoimmune thyroid disease (AITD).Since the first description in 1966by Brain et al.(Brain et al.,1966),case reports described heterogeneous clinical manifestations such as psychiatric impairments and neurological disturbances (Kothbauer-Margreiter et al.,1996).The diagnosis was established on the basis of unspecific central nervous dys-function with the presence of serum anti-thyroid autoantibodies (aAb),regardless of the thyroid disorder (Fatourechi,2005).An autoimmune pathogenic link was suggested between HE and AITD based on HE features such as (i)presence of serum anti-thyroid aAb indicating active thyroiditis (Brain et al.,1966),(ii)cerebrospinal fluid (CSF)biochemistry suggesting inflammatory process (Ferracci et al.,2003),(iii)association with other autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis (Mulhern et al.,1966),(iv)sex ratio and age distribution similar to AITD (Chaudhuri and Behan,2003;Sawka et al.,2002),(v)the improvement of symptoms after corticoste-roid therapy (Chong et al.,2003),and (vi)presence of serum aAb against neuronal alpha-enolase (Fujii et al.,2005;Ochi et al.,2002;Yoneda et al.,2007)and an unidentified 36kDa autoantigen (Oide et al.,2004).HE is also characterized by an increase in the CSF anti-thyroid,especially anti-thyroperoxidase (TPO)aAb levels (Ferracci et al.,2003)which was proposed as a valuable marker of diagnosis (Ferracci et al.,2004).

The main thyroid autoantigens,TPO and thyroglobulin (Tg),are the enzyme-substrate pair involved in thyroid hormone production (Ruf and Carayon,2006).TPO is a 933-amino-acid

Journal of Neuroimmunology 192(2007)13–

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?Corresponding author.Tel.:+33231272551;fax:+33231272550.E-mail address:blanchin-s@chu-caen.fr (S.Blanchin).

long,type I integral membrane protein,which carries out the iodination and the intramolecular coupling of tyrosine residues of Tg to form thyroid hormones.Human(Chazenbalk et al., 1993)and murine(Ruf et al.,1989)monoclonal antibodies (mAb)have been used for characterization of the TPO immuno-dominant region(IDR).

The aim of the present study was to assess the reactivity of anti-TPO aAb from HE patients with central nervous system (CNS)tissues.In immunofluorescence assays,both HE patient serum anti-TPO aAb and murine anti-TPO mAb were bound to astrocytic cells.

2.Materials and methods

2.1.HE patients and control subjects

Sera and CSF from10HE patients admitted between January 1999and April2006to the neurological ward of Caen University Hospital were collected and kept at?80°C until used.Diagnosis was based on the neurological clinical manifestations and the presence of anti-Tg and-TPO aAb in the serum and CSF in the absence of other central neurological conditions.Serum and CSF samples were also obtained from a panel of33control patients who were referred to the emergency ward with meningitis(15), intractable headache(8),peripheral neuropathy(4),multiple sclerosis(6).They were free of AITD except two who were diagnosed with an Hashimoto's thyroiditis(HT)(serum anti-TPO aAb:80and100IU/mL,normal values:b25IU/mL).Sera from 12patients with a documented HTand serum anti-TPO aAb levels ranging from90to N200IU/mL were obtained from experienced endocrinologists.Normal sera were generous gifts from4healthy adult volunteers without AITD or CNS disease.Sera from HE patients and healthy control donors were tested for the presence of anti-nuclear aAb as described(Comby et al.,2006)and of anti-onconeuronal aAb(anti-Ri,-Hu,-Yo,-CV2,-amphiphysin,-Ma2 assays,Euroimmun GmbH,Gross Gr?nau,Germany,and Ravo Diagnostika GmbH,Freiburg,Germany)according to the manufacturers'instructions.

The procedures used in this study were approved by the local institutional review committee,and informed consent was ob-tained from all donors.

2.2.ELISA for the detection of serum and CSF anti-thyroid aAb

Wells of microtiter plates(Dynatech Laboratories Inc., Chantilly,V A)were coated with phosphate-buffered saline (PBS),pH7.4,containing300ng human TPO(Biodesign Inter-national,Saco,ME)or1000ng human Tg(Valbiotech,Paris, France).After being incubated overnight at4°C,the wells were saturated with1%bovine serum albumin(BSA;Laboratoires Eurobio,Courtaboeuf,France)in PBS.They were then incubated with1/800serum dilution for anti-TPO aAb,1/400serum dilution for anti-Tg aAb or1/5dilution for CSF samples in PBS,1%BSA with0.05%Tween20for1h30min at37°C.A calibration curve was drawn up using World Health Organization standards (NIBSC Codes:66/387and65/93for anti-TPO and-Tg aAb,respectively)and the BP114as substandard plasma(The Binding Site,Saint Egreve,France).After a washing step,the anti-Tg and -TPO aAb levels were both determined using alkaline phospha-tase-coupled goat antibodies to human IgG(P.A.R.I.S,Com-piègne,France)and p-nitrophenyl phosphate(Interchim, Montlu?on,France)as substrate.Optical density(OD)was read at405nm using a Bio-Tek Elx808microplate reader(Bio-Tek Instruments Inc,Winooski,VT).Results were expressed as means of duplicate measurements in IU/mL in the case of sera and OD [ΔOD405nm=(OD405nm of coated wells)minus(OD405nm of uncoated wells)]in that of CSF.For18sera from healthy adult volunteers,threshold values for both anti-TPO and-Tg aAb was 25IU/ml,determined as mean values plus3SD from the mean.

2.3.Depletion of HE patients’sera in anti-TPO aAb

Anti-TPO aAb in sera of HE patients were depleted by performing affinity chromatography(Ruf et al.,1992).Briefly, 10mg of purified human TPO was coupled to25mL Affi-Gel15 (Bio-Rad,Marnes la Coquette,France).After extensive PBS washing,patients’sera were incubated overnight with the coupled gel at4°C under shaking.Unbound material was then checked by ELISA to ensure that it contained no anti-thyroid aAb.

2.4.Primary normal human astrocyte cell cultures

Normal primary human astrocytes derived from the whole brain of one donor,18weeks old fetus,(Cambrex Bio Science, Verviers,Belgium)were grown on coverslips in Astrocyte Basal Medium supplemented with the Astrocyte Growth Medium SingleQuots(Cambrex Bio Science).

2.5.Immunofluorescence assays

Staining was performed on cerebrum,cerebellum and thy-roid tissue sections from Macaccus rhesus monkeys(The Binding Site)and normal human astrocyte coverslips.Human astrocytes were fixed and permeabilized in100%acetone.After BSA saturation,tissues and astrocytes were incubated for1h with HE patients’sera(1/15dilution),HE patients’sera devoid of anti-TPO aAb(1/15dilution),HT patients’sera and normal sera(1/15dilution),4murine anti-TPO mAb directed towards TPO IDR:mAb47,60,15and9(Ruf et al.,1989)and1murine anti-Tg mAb:mAbJ7C9.3(Ruf et al.,1983)(1/300dilution) and with anti-glial fibrillary acid protein(GFAP)mAb(1/1000 dilution;Sigma-Aldrich,Saint Quentin Fallavier,France)in PBS,3%BSA.Dilutions of sera and antibodies yielding optimal signal/background fluorescence were experimentally determined by serial tests.Staining patterns were revealed with rhodamine or FITC coupled goat antibodies against mouse IgG (Jackson ImmunoResearch Laboratories,West Grove,PA)or FITC coupled goat antibodies against human IgG(Euroimmun GmbH)for1h.Sections were then mounted with Vectashield-DAPI(Vector Laboratories,Peterborough,UK)and viewed using an Axioskop2Plus fluorescence microscope(Carl Zeiss Vision GmbH,Munchen,Germany)with a40×oil immersion lens.Pictures were acquired with an AxioCam HRC digital

14S.Blanchin et al./Journal of Neuroimmunology192(2007)13–20

camera equipped with an AxioVision 3.2software program.The Photoshop software program (version 6;Adobe System Inc.,San Jose,CA)was used to superimpose the pictures for colocalization viewing.

2.6.SDS-PAGE and western blot analysis

Two μg of purified human TPO were incubated in Laemmli sample buffer and loaded onto 7.5%acrylamide gels.Proteins were either stained with Coomassie brilliant blue or electro-transferred onto a nitrocellulose sheet (Amersham Biosciences,Buckinghamshire,UK)for western blotting.After BSA satura-tion,the blotted membranes were incubated overnight at 4°C with HE patients'sera (1/100dilution)or mAb47(1/300dilu-tion)in PBS,0.05%Tween 20.Membranes were then incubated with alkaline phosphatase-labeled goat antibodies against human IgG or rabbit antibodies against mouse IgG (Rockland Immunochemicals for research,Gilbertsville,PA)for 2h.Finally,a revelation step was performed using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium as the appro-priate substrate (Sigma-Aldrich).2.7.Statistical analysis

Results are expressed as means ±1SD.Significance was determined using unpaired t -tests or one-way ANOVA with Dunnett's post test.Linear correlations were evaluated using R values.Significance levels were set at p b 0.01.

3.Results

3.1.Clinical and biological characteristics of HE patients The HE cohort consists of 1man and 9women.Their ages ranged from 25to 85(mean:53years)at the onset of the disease.The autoimmune thyroid disorders were HT for 8patients and Graves'disease (GD)with anti-TPO aAb for 2patients.Clinical data are presented in Table 1according to major criteria from Chaudhuri and Behan (2003)and Ferracci et al.(2004).The clinical manifestations included impaired consciousness,tremor,cephalalgia,coma,seizure,paraparesis,hemiparesis,confusion,ataxia,memory loss,myoclonus,apraxia and aphasia.Neither dementia nor behavioural disturbance was observed.In 4patients,non-migraine-type cephalalgia was initially recorded as a prominent symptom,and resolved within 2months in one while persisting for over 6months in the three others.Abnormal electroencephalography (EEG)tracing changes were observed in 3/10patients with diffuse intermittent runs of synchronous sharp waves.Brain magnetic resonance imaging (MRI)was performed in 7/10patients with normal imaging in all subjects except one with unspecific hyperintense spots in the white matter.One day to 4months after initial manifestations,9/10patients underwent a corticosteroid trial (prednisone equivalent:1mg/kg/day)during a minimum period of 6months.One patient (no.2)remained in spontaneous remission for at least 2years and did not receive corticosteroid therapy.Two months after initiation of therapy,symptoms were present in 5/9treated patients and absent in 4/9.

Table 1

Clinical features of HE patients Patient no.12345678910Sex F F F F F F F F F M Age

51413545858457552553Thyroid disorder

HT HT HT HT HT HT GD HT GD HT Symptoms

Impaired consciousness +??++++?++Tremor ++++++++++Cephalalgia

??++???++?Migraine-type headache ??????????Coma ????????+?Seizure ??+?????+?Paraparesis ??+???????Hemiparesis +?+???????Confusion ???++++?++Ataxia

+++????++?Memory loss +??????+?+Myoclonus ???+????++Apraxia ?+????????Aphasia

?+?????+++Focal neurological deficit ?

EEG Normal Diffuse abn Normal Normal Normal Normal Normal Normal Diffuse abn Diffuse abn MRI

NA Normal Normal Hypersignal NA Normal Normal Normal NA Normal Corticosteroid therapy +?++++++++Evolution at 6months

CR CR CD CR Death CD CD CR CD CR Persisting symptoms at 6months/12months

?/?

+/?

+/NA

?/NA

+/NA

+/?

+/+

?/?

F:female,M:male,HT:Hashimoto's thyroiditis,GD:Graves ’disease,+:presence,?:absence,abn:abnormalities,NA:not available,CR:complete remission,CD:corticodependence.

S.Blanchin et al./Journal of Neuroimmunology 192(2007)13–20

At6months,steroid therapy was discontinued in8/9patients. From6months to12months,corticodependence was observed in 4/9patients(no.3,6,7,9).All9steroid-treated patients were clinically improved.At12months,1(no.9)was symptomatic under therapy,5(no.1,3,4,8,10)were asymptomatic after cessation of therapy and2(no.6,7)were under therapy with a follow-up limited to7months.One patient(no.5)had died of an unrelated condition.From12months,therapy was continued in only1/10patient(no.9).All10HE patients exhibited high levels (N50IU/mL)of anti-TPO or-Tg aAb in their sera,or both (Table2).Nine HE patients were evaluated for CSF protein concentration and IgG index which were within normal or near normal ranges(Table2).No serum antibodies against nuclear and onconeuronal antigens were found in HE patients nor healthy donors(data not shown).

3.2.High frequency of anti-TPO aAb in CSF from HE patients

Thirty three CSF control samples corresponding to diverse CNS diseases with or without HT(2patients)status were quantified for their respective anti-TPO and-Tg aAb level.All these CSF exhibited lowest levels of anti-TPO and-Tg aAb which were used to determine threshold values.These values expressed inΔOD405nm(meanΔOD405nm+2SD),were set at0.228for anti-TPO aAb and0.115for anti-Tg aAb.High levels of anti-TPO (ΔOD N0.228)and-Tg(ΔOD N0.115)aAb were detected in all the HE patients'CSF(p b0.01;Fig.1).No correlation was found between serum and CSF levels of anti-TPO and-Tg aAb or between the respective anti-Tg and-TPO aAb levels.

3.3.Serum anti-TPO autoantibody titer follow-up after initiation of corticosteroid therapy

In6HE patients(no.1,2,3,4,9,10),serum anti-TPO aAb levels were evaluated2and12months after initiation of corticosteroid therapy(Fig.2).Four out of6exhibited decreased anti-TPO aAb levels after2months(p b0.01),and in3,antibody titers fell below the threshold anti-TPO aAb value (b25IU/mL)after12months.Interestingly,no alteration of anti-TPO aAb(N200IU/mL)was noted in one HE patient(no.

9)after a5-year therapy(data not shown).

3.4.Anti-TPO aAb and mAb recognize the same TPO isoforms

To determine the specificity of anti-TPO aAb from HE patients’sera,a western blotting assay was performed on affinity-purified human TPO.TPO at100kDa was detected by performing Coomassie brilliant blue staining(Fig.3,A2).In western blotting assays,anti-TPO mAb47,reacted with the2 main isoforms forming the characteristic TPO doublet around 100kDa(Fig.3,B2).Both TPO isoforms were recognized by sera from the10HE patients as shown in Fig.3,C2which depicts the typical reactivity of the HE patient's no.9serum.

3.5.Anti-TPO aAb bind to primate cerebellar tissues

To investigate more closely the role of anti-TPO aAb in CNS abnormalities,indirect immunofluorescence assays with10HE patients,12HT patients and4healthy donors’sera were performed on CNS and thyroid tissues from M.rhesus.Fig.4A depicts the typical reactivity of one of HE patients'sera(no.9), the same serum depleted from anti-TPO aAb,one HT patient serum(anti-TPO aAb N200IU/mL)and normal serum on cerebellum and thyroid tissues.The anti-TPO aAb reactivity of all sera were first evaluated by specific staining of the thyroid

Table2

Biological features of HE patients

Patient no.12345678910

CSF proteins(NR:0.15–0.45g/L)NA0.420.360.28NA0.35NA0.56NA0.69 IgG index(NR b0.7)NA0.58NA0.580.45NA NA0.47NA0.66 Serum anti-Tg aAb(NR b25IU/mL)17844N300225N30065150N30020041 Serum anti-TPO aAb(NR b25IU/mL)25150N20060N200N20080N200N200170 CSF anti-Tg aAb(ΔOD405nm b0.115)NA0.20 1.500.50 1.500.250.31 2.25 1.470.14 CSF anti-TPO aAb(ΔOD405nm b0.228)NA 3.49 2.320.280.78 2.670.75 4.03 3.91 1.89

CSF:cerebrospinal fluid,NA:not available,NR:normal range,aAb:autoantibodies,Tg:thyroglobulin,TPO:thyroperoxidase,ΔOD405nm:optical densities at 405

nm.

Fig.1.Anti-TPO and-Tg aAb in CSF from HE patients.CSF from9HE patients

was analysed by ELISA to determine the anti-TPO(A,white bars)and-Tg(B,black

bars)aAb levels.Results were expressed as the meanΔOD405nm value±1SD

(error bars).Dotted lines give threshold values for anti-TPO and-Tg aAb.

16S.Blanchin et al./Journal of Neuroimmunology192(2007)13–20

cell membrane (Fig.4,A6).The 10sera from HE patients (Fig.4,A1)but not the 12from HT patients (Fig.4,A3)or the 4from healthy subjects (Fig.4,A4)bound to structures in primate cerebellar tissues.After the depletion procedure which removed more than 90%of anti-TPO aAb but not anti-Tg aAb (Fig.4D and data not shown),the serum did not bind significantly to either thyroid cells or cerebellar cells (Fig.4,A2and A7).As the HE patients'sera contained anti-TPO and -Tg aAb,immuno-fluorescence assays were also performed with mAb directed against human TPO or Tg (Fig.4,B and C)to confirm the specificity of cerebellar staining.Four mAb characteristic of the TPO IDR (mAb47,60,15,9)and one directed toward human Tg (J7C9.3)were selected.The 4anti-TPO mAb all showed a similar reactivity pattern as seen with HE patients'sera on primate cerebellar cells (Fig.4B).Stained cells were mostly located in the subcortical cerebellar white matter,near the granular cell layer.No staining of cerebellar neuronal cells,including granular and Purkinje cells,was observed.The anti-Tg mAbJ7C9.3did not bind to cerebellar tissues (Fig.4,C1).None of the above sera and mAb bound to primate cerebrum tissues (data not shown).These results further suggest that the sole anti-TPO aAb electively recognize primate cerebellum structures.3.6.Reactivity of anti-TPO antibodies to GF AP-expressing astrocytic cells

To identify further the cerebellar cell type(s)reacting with anti-TPO aAb,a double immunofluorescence assay with HE patients'sera and anti-GFAP mAb was performed on cerebellar tissues (Fig.5).Anti-TPO aAb from 2HE patients'sera (no.2and no.3)bound to GFAP-positive astrocytic cells.Similarly,the anti-TPO mAb47stained strongly at the membrane (Fig.5,D)of normal human astrocyte primary cell cultures expressing GFAP (Fig.5,E).These data therefore indicate that anti-TPO aAb from HE patients and anti-TPO mAb both bind to astrocytes.4.Discussion

Present data suggest that anti-TPO and/or -Tg aAb are present in the CSF of HE patients and that their serum anti-TPO aAb bind cells of astrocyte lineage.

A sensitive ELISA was performed to detect CSF anti-thyroid aAb in two groups,i.e.9HE patients and 33control adults with other neurological conditions.In accordance with the HE cases already reported in the literature (Chong et al.,2003),2GD patients with subclinical or overt hyperthyroidism were included.In the HE patients,anti-TPO and/or anti-Tg aAb were detected at variable levels in sera and significant levels in CSF,consistent with previous reports (Castillo et al.,2006;Ferracci et al.,2003).Their absence in the 33control patients including 2patients with HT suggests that this finding was restricted to HE and thus may provide a clue for early HE https://www.360docs.net/doc/026499588.html,S inflammation was not documented since normal or near normal range CSF protein concentrations and IgG indices,and normal or unspecific brain MRI and EEG patterns were found.There is no evidence to conclude between either local antibody synthesis or blood brain barrier passage which is suggested in patients no.4and no.7with lowest serum and CSF aAb levels.

Anti-TPO aAb in the CNS of HE patients might contribute to CNS pathology by interacting with CNS tissues,although not excluding a role for some other pathogenic antibodies.A role of aAb is further suggested by the marked clinical

improvement

Fig.2.Time course study of serum anti-TPO aAb level in HE patients.Serum anti-TPO aAb levels (IU/ml)were sequentially determined in 6HE patients (no.1,2,3,4,9and 10)for 2to 12months after the onset of corticosteroid therapy.Results were expressed as mean values of triplicated experiments.Standard deviations from all means were lower than 10%of the mean.Threshold value for normal sera (25IU/mL)as described in the Materials and methods

section.

Fig.3.Reactivity of anti-TPO aAb from HE patients and anti-TPO mAb with purified human TPO.Purified human TPO samples were run on SDS-PAGE gel and proteins were either directly stained with Coomassie brillant blue (A,2)or electrotransferred onto a nitrocellulose membrane and revealed in western blotting experiments using either anti-TPO mAb47(B,2)or HE patient's no.9serum (C,2).The molecular weights of the standards (column 1)are indicated in kDa on the left.Data shown are of a representative experiment.

S.Blanchin et al./Journal of Neuroimmunology 192(2007)13–20

seen in 9HE patients following corticosteroid therapy which paralleled in 4/6the decrease in anti-TPO aAb level.A pathogenic role for anti-TPO aAb was also previously suspected in other conditions such as decreased intelligence quotient scores in children from mothers with high levels anti-TPO aAb and normal thyroid function during pregnancy (Pop et al.,1995)and the predisposition to depression found directly correlated with anti-TPO aAb levels (Pop et al.,1998).The complement-mediated thyrocyte cytotoxicity of anti-TPO aAb from AITD patients was found inhibited by human TPO and absent in patients with other autoimmune diseases such as lupus erythematosus and glomerulonephritis (Wa deleux et al.,1989).Antibody-dependent cell cytotoxicity against thyrocytes was dependent on anti-TPO and not anti-Tg aAb (R odien et al.,1996).A human chimeric anti-TPO Fab-dependent cell cyto-toxicity on human thyroid cells was exerted by peripheral blood mononuclear cells (Guo et al.,1997).

Due to limited CSF concentrations of anti-TPO aAb,serum anti-TPO aAb and a panel of murine anti-TPO mAb cross-reacting with HT and GD patients'sera (Ruf et al.,1989),were preferentially used for investigating further their interactions with neurological cells.Indirect immunofluorescence

experiments

Fig.4.Reactivity of anti-TPO aAb from HE patients'sera and anti-TPO mAb to primate cerebellum and thyroid tissues.Immunofluorescent stainings of human sera (A),anti-TPO mAb (B)and anti-Tg mAb (C)were performed on monkey cerebellum (A1–A5,B1–B5,C1–C2)and thyroid (A6–A10,B6–B10,C3–C4)tissues (original magnification ×400).Used antibodies are the following:one typical HE patient serum (no.9;A1,A6),the depleted HE patient no.9serum (A2,A7),one HT patient serum (A3,A8),one healthy individual serum (A4,A9),anti-TPO mAb47(B1,B6),60(B2,B7),15(B3,B8),9(B4,B9)and anti-Tg mAbJ7C9.3(C1,C4).Nucleus DAPI staining is shown in blue.Slides A-5/10,B-5/10and C-2/4exhibit non-specific binding for each tissue when no first antibody was added.Panel D depicts the results of a representative experiment where the HE patient no.9serum were depleted from anti-TPO aAb.Results are expressed as the binding rate to coated human TPO±1SD (error bars).

18S.Blanchin et al./Journal of Neuroimmunology 192(2007)13–20

exhibited specific staining of cerebellar cells from M.rhesus monkeys while none using HT patients'and healthy donors'sera.The reactivity of HE aAb with TPO epitopes was ascertained by the similar binding pattern of 4anti-TPO mAb on the same cells and confirmed in western blotting experiments showing that HE patients'sera and mAb47,an anti-TPO IDR mAb bound the same TPO isoforms forming a doublet around 100kDa (Ruf and Carayon,2006).Anti-TPO aAb depletion from HE patients'sera confirmed that the anti-TPO aAb bound solely to CNS structures.Furthermore,no cerebellar binding was detected with anti-Tg mAbJ7C9.3,which do not cross-react with anti-TPO aAb (Ruf et al.,1992).Serum aAb from HT patients did not bind cerebellar tissue consistent with differences in anti-TPO aAb repertoire between HT and HE patients.Localization of the stained cells in the subcortical cerebellar white matter and their typical star-shaped morphology were consistent with their astrocytic lineage.In double immunostaining experiments,binding of anti-TPO aAb from HE patients was colocalized with GFAP,an astrocytic marker.This was further supported by binding of the anti-TPO mAb47to normal human astrocytes in primary cultures.In-terestingly,a strong relationship between HTand cerebellar ataxia was reported (Selim and Drachman,2001).Moreover,mild astrocytic gliosis and microglial activation were previously mentioned in reports based on brain biopsies from HE patients (Castillo et al.,2006;Nolte et al.,2000).An involvement of astrocytes was noted in other CNS dysfunctions such as epilepsy and Alzheimer's disease (Blasko et al.,2004;Schipper,1996).Cephalalgia observed in several of the present HE patients may reveal astrocyte-related spreading depression (Chuquet et al.,2007).

That aAb trigger neuropathic mechanisms has been previ-ously documented for anti-glutamic acid decarboxylase aAb and anti-calcium channel aAb in patients with the Stiff-person

syndrome (Dalakas et al.,2000)and the Lambert –Eaton myasthenic syndrome,respectively (Rosenfeld et al.,1993).In the present study,presence of serum and CSF anti-TPO aAb,binding of anti-TPO aAb on astrocytic cells,and improvement of the clinical symptoms after reducing the level of serum anti-TPO aAb partially meet the criteria for demonstrating patho-genicity of aAb (Sutton and Winer,2002).Although not excluding the role of other neuropathogenic antibody(ies)in HE,present data prompt identification of the astrocytic anti-genic target(s)of HE anti-TPO aAb.Experimental animal studies are needed to investigate whether passive anti-TPO aAb transfer reproduces the main features of the disease and im-munization with the putative antigen(s)leads to an animal model of the disease providing clues how neurological func-tions are impaired.Acknowledgments

This research was supported by a grant from the french ministère de la santéet de la protection sociale,programme hos-pitalier G42006:“Lymphocytes B et ses effecteurs normaux et pathologiques ”.We thank Dr.Benoit Dupuy and Dr.Nizam Kassis,h?pital Louis Pasteur Cherbourg,for providing sera and CSF from HE patient.The authors are also grateful to Pr.Fran?oise Chapon,laboratoire de neuropathologie CHU Caen,for helpful advices.References

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Fig.5.Immunoreactivity of anti-TPO aAb and mAb47to GFAP-specific astrocytic cells.Immunofluorescent staining patterns were obtained on monkey cerebellum (A –C)with sera from HE patients no.2and no.3(A –B,in green)and anti-GFAP mAb (A –B,in red)and on normal human astrocyte primary cultures (D –F)with anti-TPO mAb47(D,in red)and anti-GFAP mAb (E,in red).Nuclei are shown in blue.Non-specific binding are shown in slides C and F.(Original magnification ×400).

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