Patent Application: US-201615192064-A

Abstract:
the serotonin receptor 5ht2a and membrane nadph oxidases are found to be a target of autoantibodies present in multiple sclerosis patients . the present invention refers to peptides comprised in the extracellular regions of the human 5ht2ar and / or noxs for diagnosis and therapy of multiple sclerosis .

Description:
in the study ms group comprises men and women between 15 and 50 years of age who meet all the following criteria : diagnosis of relapsing / remitting ms , according to mcdonald criteria ; an expanded disability scale score ( edss ) between o and 5 . 0 ; lesions detected by mri compatible with the diagnosis of multiple sclerosis ; at least one acute episode in the last 12 months . control samples include other neurological disorders affected patients ( including inflammatory , degenerative diseases not involving direct or indirect de - myelinization , i . e . : cerebral cancers , stroke , vasculitis , etc ) that need differential diagnosis with multiple sclerosis . control patients were selected by sex and age to be similar to multiple sclerosis patients . blood serum was collected , from each patients . from the blood serum , to perform the experiments on cell culture , the igg fractions were purified . patients gave written informed consent before any study - related procedures was performed . the purification of igg fractions from serum of ms and control neurological subjects has been carried out by affinity chromatography on a / g sepharose columns ( pierce , rockford , ill .). the protein concentration of immunoglobulin fractions has been assessed spectrophotometrically . m03 - 13 cells — the m03 - 13 cells are an immortal human - human hybrid cell line with the phenotypic characteristics of primary oligodendrocytes ( ols ), derived from the fusion of a 6 - thioguanine - resistant mutant of a human rhabdomyosarcoma with ols obtained from adult human brain ( cellution biosystem inc ., canada ). they were grown in dulbecco &# 39 ; s modified eagles medium ( dmem ; gibco invitrogen ), containing 4 . 5 g / l glucose ( gibco , auckland , new zealand ), supplemented with 10 % foetal bovine serum , 100 u / ml penicillin and 100 μg / ml streptomycin ( fbs ; sigma s . louis , usa ). hek293 cells — hek293 is a cell line derived from human embryonic kidney cells ( american type culture collection , atcc , usa ). they were grown in dulbecco &# 39 ; s modified eagles medium ( dmem ; gibco invitrogen ), containing 4 . 5 g / l glucose ( gibco , auckland , new zealand ), supplemented with 10 % fbs , 100 u / ml penicillin and 100 μg / ml ( sigma s . louis , usa ). hela and sh - sy5y cells — the human cervical adenocarcinoma hela cells and the human neuroblastoma sh - sy5y cell lines ( american type culture collection , atcc , usa ) were grown in dmem - f12 medium ( gibco invitrogen ) containing 4 . 5 g / l glucose ( gibco , auckland , new zealand ), supplemented with 10 % fbs ( sigma s . louis , usa ), 100 u / ml penicillin and 100 μg / ml the cells were kept in a 5 % co2 and 95 % air atmosphere at 37 ° c . m03 - 13 cells were grown to semiconfluency in 60 - mm culture dishes . after trypsin detachment , 5 · 10 5 cells were suspended in 1 ml of phosphate buffered saline ( pbs ) and fixed overnight with 1 % formaldehyde at room temperature . next , cells were permeabilized with 0 . 1 % triton x - 100 for 40 min at 4 ° c ., washed 4 × with 2 ml of pbs containing 2 % fbs , 0 . 01 % nan3 , 0 . 1 % triton x - 100 ( buffer a ), and incubated for 45 min at 4 ° c . with 1 : 50 dilution of rabbit polyclonal to 5ht2ar antibody ( abcam ab81864 ). the cells were then washed twice with the same buffer and incubated for 45 min at 4 ° c . with cy3 - conjugated anti -( rabbit igg ) ig ( amersham pharmacia biotech ) at 1 : 50 dilution . control cells were incubated with cy3 - conjugated anti -( rabbit igg ) igg alone . after two washes in buffer a , cells were resuspended in pbs and analyzed by flow cytometry using facscan ( bd , heidelberg , germany ) and winmdi software . hek293 cells were plated in 100 mm petri dishes and grown to semiconfluence . after trypsinization and wash in pbs , the cells were resuspended in 200 μl pbs and then incubated with mouse serum for 30 min at 4 ° c ., to block nonspecific binding ; then , they were incubated for 30 min with 200 μg of serum igg ( ms or neurological ), and stained for 30 min with pe - conjugated goat anti human igg . cells were washed and resuspended in 200 μl of pbs for flow cytometric analysis of phycoerythrin positive cells with a facsscan apparatus ( becton - dickinson ). data were analyzed using winmdi software . mo3 - 13 cells , grown to semiconfluence in 100 mm dishes , were incubated for 18 h in 0 . 2 % fbs medium . the cells were washed twice with pbs and harvested in cold ripa buffer containing 2 . 5 mm na - pyrophosphate , 1 mm r - glycerophosphate , 1 mm navo4 , 1 mm naf , 0 . 5 mm pmsf , and the cocktail of protease inhibitors . the cells were kept for 15 min at 4 ° c . and disrupted by repeated aspiration through a 21 - gauge needle . cellular debris was pelleted by centrifugation at 11600 g for 15 min at 4 ° c . 300 μg of cellular lysates were immunoprecipitated with igg from neurological or ms patients at 1 : 10 dilution . samples were rocked gently for 16 h ; thereafter 20 μl of protein a / g plus - agarose ( santa cruz biotechnology ), resuspended in ripa buffer , was added to immunoprecipitates . samples were further rocked for 1 h , centrifuged at 3000 rpm . supernatants were collected and the protein a / g plus - agarose was added again , to obtained the immunodepleted samples . then , the pellets were washed thrice in ripa buffer and once with pbs before the addition of 20 μl laemmli sample buffer . immunoprecipitated / immunodepleted samples and 50 μg of total lysates in laemmli buffer were boiled for 5 min and centrifuged for 1 min at 11600 g at room temperature ( 22 ° c .). the pellets were discarded and supernatants were resolved by 7 . 5 % sds - page and transferred onto nitrocellulose membrane . next , the membrane was blocked in 3 % dry - fat milk in tbs - tween20 ( 0 . 05 %) and probed with a polyclonal anti - human anti nox3 ( abcam ab81864 ) or 5ht - 2a receptor ( abcam ab85496 ) antibodies at 1 : 1000 dilution . then , the membrane was washed and incubated with a secondary horseradish peroxidase - linked antibody ( amersham pharmacia biotech ) 1 : 2000 and was detected by ecl . diluted peptide ( 20 μg / ml ) was coated to the wells of a pvc microtiter plate and incubated at 4 ° c . overnight . after many wash , the remaining protein - binding sites was blocked with 3 % bsa solution and incubated at 4 ° c . overnight . the patient extracted immunoglobulins was diluted in blocking buffer and incubated at 4 ° c . overnight . the plate was washed for four times with pbs . for the detection , we used a secondary antibody anti human ( recognized constant region of the patient antibody conjugated with hrp and ( 3 , 3 ′, 5 , 5 ′- tetramethylbenzidine ) tmb solution that was added for each well and incubated for 15 - 30 min . equal volume of stopping solution ( 2 m h2so4 ) was added to the plate and absorbance ( optical density ) of plate was read at 450 nm . beads linked peptide ( 6 × 10 4 beads / sample ) were mixed with different concentrations of patient extracted immunoglobulins for 16 h at 4 ° c . in a rotator . the beads were washed twice with pbs by centrifugation at 14 , 000 rpm for 2 min at room temperature and were resuspended in 100 μl of pbs . for the detection , we used a secondary antibody anti human ( recognized constant region of the patient antibody conjugated with hrp and ( 3 , 3 ′, 5 , 5 ′- tetramethylbenzidine ) tmb solution that was added for each well and incubated for 15 - 30 min . equal volume of stopping solution ( 2 m h2so4 ) was added to the plate and absorbance ( optical density ) of plate was read at 450 nm . detection of peptide / immunoglobulins derived from patients interaction on beads by flow cytometry beads linked peptide ( 6 × 10 4 beads / sample ) were mixed with different concentrations of patient extracted immunoglobulins for 16 h at 4 ° c . in a rotator . the beads were washed twice with pbs by centrifugation at 14 , 000 rpm for 2 min at room temperature and were resuspended in 100 μl of pbs . then , the samples was incubated with anti - human secondary antibodies conjugated with fitch and beads were analyzed on a bd facs calibur ( becton - dick - inson , franklin lakes , n . j . ), and the data analyzed on flowjo ( treestar , ashland , oreg .) software . ros levels were determined using the membrane - permeant fluorogenic probe 5 , 6 - carboxy - 2 ′, 7 ′- dichlorofluoresceindiacetate , dchfda ( molecular probes , leiden , the netherlands ). the assay was based on the fluorescence detection of dichlorofluorescein ( dcf ), formed by ros - mediated oxidation of the non - fluorescent precursor , dichlorofluorescin . mo3 - 13 cells were grown to semi - confluence in 24 multiwell plates ( 45000 cell / well ) and incubated for 18 h in medium containing 0 . 2 % fbs before the experiments . the cells were washed twice with fbs free medium and incubated with 50 μm of ddsk for 30 min at 37 ° c . and then with 200 μg / ml of igg from ctr or ms patients ( damiano et al ., 2013 ) for 30 min at 37 ° c . the cells were incubated with 10 μm dchf - da for 10 min and washed three times with pbs containing 10 mm glucose , 1 . 2 mm mgcl 2 and 1 . 2 mm cacl 2 . dcf fluorescence was measured using the plate reader fluoroskan ascent fl fluorometer ( thermo electron oy , vantaa , finland ) and data analyzed by ascent software . to evaluate the effects of 5ht on ros levels , a dose of 50 μm of the substance was added to the cells after dchf - da incubation and dcf fluorescence was measured at different time intervals . total rna was extracted using trizol reagent according to the protocol provided by the manufacturer ( sigma - aldrich ). total rna ( 1 μg ) was reverse transcribed using transcriptor first strand cdna syn - thesis kit ( roche applied science , monza , italy ) by oligo - dt primers for 30 min at 55 ° c . in a 20 μl reaction volume . semi - quantitative pcr was performed using hot master taqdna polymerase ( 5prime ) in 20 μl final volume containing 0 . 2 mm dntp , 0 . 2 μm of the specific primers and 100 ng of sample cdna . the pcr conditions used were 94 ° c . 2 min , ( 94 ° c . 30 s , 60 ° c . 30 s , 70 ° c . 30 s ) and 70 ° c . 5 min . the reactions were carried out at 35 number of cycles . h - 5ht2ar / egfp construct : h - 5ht2ar gene ( ncbi accession number : np_000612 ) has been inserted in the pegfp - n3 vector from clontech . the cdna is cloned between bamhi and bgiii sites in the mcs . the cells were transfected with h - 5ht2ar / egfp construct . one day before transfection , 450 . 000 cells ( hek293 ) were plated in 35 mm dishes in growth medium so that cells will be 70 - 90 % confluent at the time of transfection . for each transfection sample , complexes were prepared as follows : 1 γ / λ dna ( 5 - ht2a receptor conjugated to egfp ) was added to 80 μl of growth medium without serum and antibiotics . 4 μl of lipofectamine ™ 2000 was added to 80 μl of growth medium without serum and antibiotics . the diluted dna was combined with diluted lipofectamine ™ 2000 , mixed gently and incubated for 45 minutes at room temperature . then the complexes were added to cells . cells were incubated at 37 ° c . in a co2 incubator for 18 - 48 hours prior to testing . antibodies . duox 1 and 2 proteins were detected with a rabbit polyclonal antibody raised against the peptide sequence eteltpqrlqc located inside the first intracellular loop of human duox1 ( damiano et al ., plosone ). p - erk1 / 2 ( sc - 7383 ) and haras ( sc - 520 ) antibodies were purchased by santa cruz ; nox3 ( ab81864 ) and h - 5ht2ar antibodies ( ab85496 ) were purchased by abcam . total cells lysates were obtained in ripa buffer ( 50 mm tris - hcl , ph 7 . 5 , nacl 150 mm , 1 % np40 , 0 . 5 % deoxycholate , 0 . 1 % sds ) containing 2 . 5 mm na - pyrophosphate , 1 mm β - glycerophosphate , 1 mm navo4 , 1 mm naf , 0 . 5 mm pmsf , and a cocktail of protease inhibitors ( roche , usa ). the cells were kept for 15 min at 4 ° c . and disrupted by repeated aspiration through a 21 - gauge needle . cell lysates were centrifuged for 10 min at 13000 rpm and the pellets were discarded . fifty micrograms of total proteins were subjected to sds - page under reducing conditions . after electrophoresis , the proteins were transferred onto a nitrocellulose filter membrane ( bio - rad laboratories , uk ) with a trans - blot cell ( bio - rad laboratories , uk ) and transfer buffer containing 25 mm tris , 192 mm glycine , 20 % methanol . membranes were placed in 5 % non - fat milk in phosphate - buffered saline , 0 . 5 % tween 20 ( tbst ) at 4 ° c . for 2 h to block the nonspecific binding sites . filters were incubated with specific antibodies before being washed three times in tbst and then incubated with a peroxidase - conjugated secondary antibody ( santa cruz ). after washing with tbst , peroxidase activity was detected with the ecl system ( ge - healthcare , uk ). the filters were also probed with an anti α - tubulin antibody ( sigma , usa ). protein bands were revealed by ecl and , when specified , quantified by densitometry using image ) software . densitometric values were normalized to α - tubulin . since , depending on the cell type or tissue , bands of different sizes can appear by western blotting for 5ht2ar ( fragments or protein complexes ), to determine which bands are specific , before proceeding with the staining protocol , the antibody was incubated with an excess of peptide that correspond to the epitope recognized by the antibody . by comparing the staining from the blocking antibody vs the antibody alone it has been possible to evidence the specific 5ht2ar staining . the linear and clips peptides are synthesized based on the amino acid sequence of the target protein using standard fmoc - chemistry and deprotected using trifluoric acid with scavengers . the constrained peptides are synthesized on chemical scaffolds in order to reconstruct conformational epitopes , using chemically linked peptides on scaffolds ( clips ) technology ( timmerman et al . ( 2007 ). for example , the single looped peptides are synthesized containing a dicysteine , which was cyclized by treating with alpha , alpha ′- dibromoxylene and the size of the loop is varied by introducing cysteine residues at variable spacing . if other cysteines besides the newly introduced cysteines are present , they are replaced by alanine . the side - chains of the multiple cysteines in the peptides are coupled to clips templates by reacting onto credit - card format polypropylene pepscan cards ( 455 peptide formats / card ) with a 0 . 5 mm solution of clips template such as 1 , 3 - bis ( bromomethyl ) benzene in ammonium bicarbonate ( 20 mm , ph 7 . 9 )/ acetonitrile ( 1 : 1 ( v / v )). the cards are gently shaken in the solution for 30 to 60 minutes while completely covered in solution . finally , the cards are washed extensively with excess of h 2 o and sonicated in distrupt - buffer containing 1 percent sds / 0 . 1 percent beta - mercaptoethanol in pbs ( ph 7 . 2 ) at 70 ° c . for 30 minutes , followed by sonication in h 2 o for another 45 minutes . the binding of antibody to each peptide is tested in a pepscan - based elisa . the 455 - well credit card format polypropylene cards containing the covalently linked peptides are incubated with primary antibody solution for example consisting of 1 / 1000 diluted serum in blocking solution , for example 4 % horse serum , 5 % ovalbumin ( w / v ) in pbs / 1 % tween . after washing , the peptides are incubated with a 1 / 1000 dilution of antibody peroxidase conjugate for one hour at 25 ° c . after washing , the peroxidase substrate 2 , 2 ′- azino - di - 3 - ethylbenzthiazoline sulfonate ( abts ) and 2 microlitres of 3 percent h 2 o 2 are added . after one hour , the color development are measured . the color development are quantified with a charge coupled device ( ccd )— camera and an image processing system ( slootstra et al ., 1996 ). the raw data are optical values obtained by a ccd - camera calibrated to export absorption values compatible with a standard 96 - well plate elisa - reader . first the ccd - camera makes a picture of the card before peroxidase coloring and then again a picture after the peroxidase coloring . these two pictures are subtracted from each other yielding a binding value for each peptide . this data is entered in the peplab ™ database for secure storage and retrieval . all raw data are provided in an excel file and the full technical report include plots of the binding activity to all peptides and a 3d visualization of all epitope candidates identified . methods are described in timmerman et al . ( 2007 ). functional reconstruction and synthetic mimicry of a conformational epitope using clips &# 39 ; technology ). structural aspects of antibody - antigen interaction revealed through small random peptide libraries ( slootstra et al ., 1996 ). primary cultures of cortical opcs and neurons are prepared as described by cheli et al . ( 2015 ). for opcs preparation , cerebral hemispheres from 1 day old mice are mechanically dissociated and plated on poly - d - lysine - coated flasks in dulbecco &# 39 ; s modified eagle &# 39 ; s medium and ham &# 39 ; s f12 ( 1 : 1 v / v ), containing 100 μg / ml gentamicin and supplemented with 4 mg / ml dextrose anhydrous , 3 . 75 mg / ml hepes buffer , 2 . 4 mg / ml sodium bicarbonate and 10 % fetal bovine serum ( fbs ). after 24 h the medium is changed and the cells are grown in dmem / f12 supplemented with insulin ( 5 μg / ml ), human transferrin ( 50 μg / ml ), sodium selenite ( 30 nm ), d - biotin ( 10 mm ), 0 . 1 % bsa ( sigma ), 1 % fbs and 1 % horse serum . after 9 days , opcs are purified from the mixed glial culture by the differential shaking and adhesion procedure and allowed to grow on poly - dlysine - coated coverslips in culture media plus pdgf ( 10 ng / ml ) and bfgf ( 10 ng / ml ). opcs are kept in mitogens ( pdgf and bfgf ) for 2 days and then induced to exit from the cell cycle and differentiate by switching the cells to a mitogen - free medium ( mn2 ). cortical neurons are prepared from the brains of 1 - to 2 - day - old mouse . brain cortices are isolated and dissociated by digestion with a solution of 0 . 05 % trypsin ( sigma ) containing dnase i ( 0 . 06 %) in neurobasal medium for 10 min at 37 ° c . the digestion reaction is stopped with neurobasal medium containing 10 % fetal bovine serum and triturated by repeated passages ( 20 times ) through a 10 ml pipette . the cell suspension is filtered through a sterile cell strainer ( 70 μm ) into a 50 ml centrifuge tube . the cells are pelleted by centrifugation at 200 g for 5 min , and resuspended in neurobasal medium plus 2 % ( v / v ) b27 supplemented with 0 . 25 mm glutamax i , 0 . 25 mm glutamine ( invitrogen ), and 100 μg / ml gentamicin . high - density cultures ( 5 × 105 cells , ˜ 2500 cells / mm2 ) are plated onto 20 mm 2 tissue culture wells coated with poly - d - lysine . the neurons are kept at 37 ° c . in 95 % air 5 % co2 for 7 days in vitro and used for co - cultures . after 7 days in vitro , cortical neuron cultures consist of neurons essentially free from non - neural cells . co - cultures are prepared by the addition of opcs to the cultures of cortical neurons at a density of 3 × 105 cells / ml . these cultures are maintained in dmem / f12 , 1 % fbs for 7 and 14 days ( cheli et al ., 2015 ). the neuron - opc co - colture model allows the evaluation of the myelination stage of mature ol , by confocal microscopy categorizing them in three different stages : ( 1 ) cells that only extend processes but do not contact with neurofilaments ; ( 2 ) cells that establish contact with neurofilaments but do not myelinate ; and ( 3 ) cells that wrap axons and have at least two internodes connected to the cell body ( barateiro and fernandes , 2014 ). m03 - 13 cell migration is assessed by in vitro scratch assay , based on the creation of an artificial gap on a confluent cell monolayer . m03 - 13 cells are grown in complete medium and when cells reach 70 - 80 % confluence , a wound is made across the cell layer using a cell scraper . then cultures are washed in complete medium and are allowed to migrate for 24 - 48 h and the number of cells that moved across the injury line is counted at microscope . intracellular ca 2 + levels are measured fluorimetrically using the membrane - permeable ca 2 + - sensitive dye fluo - 3 - am . briefly , cells are washed twice with us buffer ( 137 mn nacl , 2 . 7 mm kcl , 1 . 0 mm mgcl 2 , 1 . 8 mm cacl 2 , 0 . 2 mm nah 2 po 4 , 12 mm nahco 3 , 5 . 5 mm glucose , ph 7 . 4 ) and incubated with 10 μm fluo - 3 - am and 0 . 02 % pluronic acid for 1 h at room temperature in the dark . cells are then washed twice with us , before adding the substances to be tested and the changes in fluorescence is measured at different time intervals using fluoroskan ascent fluorescent plate reader ( thermoelectron oy , vantaa , finland ) and data analyzed by ascent software . induction of eae ( experimental autoimmune encephalomyelitis ) as murine model for human multiple sclerosis mice will be immunized subcutaneously with 100 ml of emulsified incomplete freund &# 39 ; s adjuvant supplemented with 500 mg of mycobacterium tuberculosis h37ra ( difco ) and 100 mg of mog35 - 55 , and will receive an intraperitoneal injection of 200 ng of pertussis toxin ( list biological laboratories ) at the time of immunization and 48 hours later . the mice will be observed daily for clinical signs and scored as described ( shi et al ., 2011 ). mice will be euthanized , and brains and spinal cords will be removed and fixed by immersion with a 10 % neutral - buffered formalin solution and decalcified . fixed tissues will be embedded in paraffin , sectioned , and stained with h & amp ; e and serial histological sections will be stained also immunohistochemically to determine the distribution and types of inflammatory cells in the brain and spinal cord and the demyelination . spinal cord pathology will be assigned by scores with an experienced pathologist as described previously ( shi et al ., 2011 ) 2 - 4 × 10 6 cells are resuspend in 100 μl 1 × annexin v binding buffer . 5 μl annexin v alexa fluor 488 was added to the samples and incubated in the dark for 15 minutes at room temperature . 100 μl of 1 × annexin v binding buffer and 4 μl of pi at final pi concentration of 2 μg / ml are added to each reaction tube . the samples are incubated in the dark for 15 minutes at room temperature . the cells are washed with 500 μl 1 × annexin v binding buffer . samples centrifuged at 335 × g for 10 minutes and supernatant decanted . cells are resuspended in 500 μl 1 × annexin v binding buffer and 500 μl 2 % formaldehyde to create a 1 % formaldehyde ( fixative ) solution . tubes are mixed by gentle flicking and fixed on ice for 10 minutes . the cells are washed with pbs and diluted with rnase a at the final concentration of 50 μg / ml and incubated for 15 min at 37 ° c . the tubes are centrifuged at 425 × g for eight minutes and samples are analyzed by cytofluorimetry . statistical differences were evaluated using a student &# 39 ; s t - test for unpaired samples . 5 - ht2ar protein expression in m03 - 13 cells was evaluated by indirect immunofluorescence and flow cytometry ( fig1 a ) and by western blotting analysis ( fig1 b ). as can be shown in the figure , m03 - 13 cells express 5ht2a receptor protein . since , depending on the cell line , 5ht2a receptor protein can appear as fragments of the full antigen or a complex containing the antigen , with different molecular weights , inventors performed western blotting experiments preincubating the primary antibody in the absence or presence of a tow fold excess of immunizing peptide ; in the presence of the peptide , the specific bands disappear or are attenuated . as shown by the immunized blocking peptide experiment ( fig1 b , right panel ), in m03 - 13 cells the 5ht2ar appears as a double band of 20 - 30 kd . the pcr analysis of human 5ht2ar ( fig1 c ) shows that m03 - 13 cells , and not the human embryonic kidney cell line hek293 , express 5ht2ar mrna . to evaluate the hypothesis of the presence of igg directed against 5ht2ar in the serum from ms patients , m03 - 13 cells were immunoprecipitated with igg from control and ms subjects and then immunoblotted with anti h - 5ht2ar antibody . as shown in fig2 , multiple sclerosis igg immunoprecipitated 5ht2ar protein demonstrating a direct interaction between serum igg ms and the receptor protein . to confirm the direct interaction between igg ms and 5 - ht2ar , we performed flow cytometric surface binding experiments on hek293 cells transfected in transient with the h - 5ht2ar / egfp construct . neither the igg from control subjects ( ign ), nor those from ms patients ( igg ms ) significantly bound to the surface of mock transfected cells ( fig3 , upper panel ). in cells transfected with h - 5ht2ar / egfp construct ( lower panel ), the binding of igg to transfected cells , calculated on gfp positive cell population , was 26 % for ign , and 77 % for igms . this experiment indicates , therefore , the existence of a specific binding of the igg ms at the cell surface of the hek293 cells transfected with 5ht2ar . as shown in fig4 , m03 - 13 cells express the nox enzyme family members nox3 and nox5 isoforms ( fig4 a ). the inventors focused their attention on nox3 since this isoform shows a higher percentage of identity ( 58 . 8 ) with nox2 than nox5 ( 32 . 6 %). nox2 is expressed in oligodendrocytes in vivo ( cavaliere et al ., 2013 ). in particular , nox3 extracellular domains show a certain degree of identity with nox2 extracellular domains . this is not the case for nox5 . the inventors first evaluated whether nox3 directly interacts with 5ht2ar by immunoprecipitation of cell extract with anti h - nox3 antibody followed by western blotting with anti h - 5ht2ar antibody . as shown in fig4 b , anti nox3 antibodies immunoprecipitated 5ht2ar protein . the receptor staining was significantly decreased in the immunodepleted sample . the inventors further evaluated whether igg from ms patients interact with nox3 in mo3 - 13 cells . to this aim m03 - 13 cells were immunoprecipitated with igg from control and ms subjects and then immunoblotted with anti h - nox3 antibody . as shown in fig5 , igms immunoprecipitated nox3 protein demonstrating a direct interaction between serum igg from ms patients and nox3 . overall , our data suggest that iggs from ms patients bind to 5ht2ar and to nox3 . since nox3 binds to 5ht2ar , it is possible that a membrane protein complex constituted by 5ht2ar , nox3 and the iggs from ms is present in m03 - 13 cells and in vivo . precision epitope mapping of nox2 and 5 - ht2a receptor extracellular domains with the serum from multiple sclerosis affected patients . approximately 1250 different peptides including linear and clips peptides have been designed and synthesize based on nox2 heavy chain amino acid sequence ( seq id 1 ) and other 1250 peptides were designed and synthesize based on the human 5 - ht2a receptor amino acid sequence ( seq id 2 ). in particular , in both cases , only the extracellular domains of the proteins have been used for the design of the two peptide libraries . for nox2 were selected the regions 30 - 48 ( seq id 3 ), 124 - 169 ( seq id 4 ) and 222 - 261 ( seq id 5 ) while for 5 - ht2ar were selected the regions 1 - 76 ( seq id 6 ), 133 - 148 ( seq id 7 ), 215 - 233 ( seq id 8 ), 347 - 362 ( seq id 9 ). all peptides have been synthesized in a peptide array format and the binding of 20 different sera to all peptide libraries were measured in an elisa based set up following the procedure described in detail in the method section “ peptide synthesis and screening assays ”. in particular 2 sets of experiments were performed on the 2 libraries by using 2 different dilutions of the human patients sera . in the first set of experiment the 20 sera were used at 1 : 2500 dilutions , while in the second experiment the dilution of the sera was 1 : 1000 . the binding of the different sera to all peptides was quantified and analyzed in detail ( tables 1 and 2 , fig1 and 16 ). htr . mat : double looped conformational peptide , htr . lin15aa : linear peptide of 15 amino acids , htr . lin9 : linear peptide of 9 amino acids , htr . p2_15aa : single looped conformational peptide of 15 amino acids . in double looped peptides , three cysteine residues were added , two as first and last amino acid and one in the middle of the sequence . then peptides of the invention may the whole sequence or the fragments located between two cysteine residues or the sequence with only one cysteine at either end of the sequence . in single looped peptides , two cysteine residues were added , as first and last amino acid . then peptides of the invention may the whole sequence or the fragment located between two cysteine residues or the sequence with only one cysteine at either end of the sequence . cb245 . mat : double looped conformational peptide , cb245 . lin15aa : linear peptide of 15 amino acids , cb245 . lin9 : linear peptide of 9 amino acids , cb245 . p2_15aa : single looped conformational peptide of 15 amino acids . in double looped peptides , three cysteine residues were added , two as first and last amino acid and one in the middle of the sequence . then peptides of the invention may the whole sequence or the fragments located between two cysteine residues . in single looped peptides , two cysteine residues were added , as first and last amino acid . then peptides of the invention may the whole sequence or the fragment located between two cysteine residues or the sequence with only one cysteine at either end of the sequence . fig1 reports the alignments of the peptides of the invention with fragments of 5ht2ar . it shows that each extracellular region of the 5ht2a receptor overlap with peptides that have an high affinity for ms sera . the asterisks show the 100 % identity of amino acids with the different extracellular region of the 5ht2a receptor . fig1 reports the alignments of the peptides of the invention with fragments of nox2 . it shows that the extracellular loop 2 and loop 3 of nox 2 overlap with peptides that have an high affinity for ms sera . the asterisks show the 100 % identity of amino acids with the different extracellular region of nox2 loop 2 ( panel a ), loop 3 ( panel b ). dose - response curves of the interaction between ms igg and ddsk peptide the inventors have demonstrated the immunoglobulins of multiple sclerosis patients bind serotonin receptor . to evaluate the peptide concentration to use for elisa assay we performed a dose - response curve . the immunoglobulins of multiple sclerosis patients and control ( 200 μg ) were incubated with different concentrations ( 50 - 100 - 250 μm ) of peptide ddskvfkegs ( named ddsk in the figures ) and the absorbance ( optical density ) of plate was read at 450 nm . fig6 shows that ms igg bind the ddsk peptide at the concentration higher than 50 μm . the ms igg binds peptides derived from serotonin receptor . to confirm this data , the inventors performed indirect elisa using two different peptides derived from serotonin receptor compared to scrambled . the immunoglobulins of multiple sclerosis patients and control ( 200 μg ) were incubated with 100 μm of specific or control peptide and the absorbance ( optical density ) of plate was read at 450 nm . fig7 shows that ms igg bind specifically ddskvfkegs (( seq id no : 157 ), also named ddsk ) and lygyrwplpskl (( seq id no : 158 ), also named lygy ) peptides respect to scrambled ( twyahncrlq , seq id no : 173 ). the inventors have demonstrate the ms igg binds specific peptide derived from receptor . to evaluate the sensitivity of the binding , the inventors performed a roc curve starting from 28 samples each one repeated at least 4 times . in fig8 we show that binding between ms igg and ddsk peptides has a 82 % of sensitivity and 96 % of specificity . to evaluate whether the interaction of igg from ms patients with the membrane 5ht2ar / nox3 complex could affect cell signaling downstream the receptor , the inventors performed experiments with hek293 cells transfected in transient with 5ht2ar . the cells were stimulated with igg from control or ms patients prior to the stimulation with the endogenous receptor agonist 5 - ht , and then p - erk1 / 2 levels were measured . as shown in the fig9 a , igg from ms patients inhibit 5 - ht - mediated induction of p - erk1 / 2 levels . the inventors also provided evidences about the interference of igg from ms patients with 5ht2ar signaling in human oligodendrocyte cell line m03 - 13 . cells were stimulated with igg from control or ms patients in the presence and absence of risperidone , a serotonin receptor antagonist . in the presence of the substance , igg from ms patients failed to induce p - erk1 / 2 levels thus confirming that autoantibodies present in sera of ms patients increase p - erk1 / 2 levels acting on serotonin receptor ( fig9 b ). overall , the present results suggest that 5ht2ar - bound autoantibodies present in ms patients may exert an ethiopathogenic role in ms . therefore , compounds interfering with the immunoglobulins - receptor binding can be used for the treatment of ms . igg from ms patients increase reactive oxygen species ( ros ), duox1 / 2 , p - erk1 / 2 , haras and nox3 protein levels and ddsk peptide reverts the effect 5ht receptors rely on ros for downstream signaling ( kruk et al ., 2013 ). therefore , we measured ros levels , as dcf fluorescence , in m03 - 13 cells stimulated with igg from control or ms patients in the absence or presence of the ddsk peptide . as shown in fig1 a , igg ms significantly increased ros levels and preincubation of cells with ddsk peptide reverted the effect . on the contrary , ddsk did not significantly affect ros levels of iggctr treated samples . scrambled peptides , used as negative control , did not influence ros levels of igg ms treated samples ( fig1 b ). the ability of ddsk to interfere with 5ht2a receptor downstream signaling measured as ros levels , demonstrate that the peptide is able to revert the biological effects of the autoantibodies present in ms sera on human oligodendrocytes and , therefore , that it may be used for the treatment of multiple sclerosis . in addition to nox3 and nox5 , m03 - 13 cells express also duox1 and 2 isoforms ( damiano et al ., 2012 , plos ); their protein levels are very sensitive to ros . other downstream ros targets are p - erk , and h - ras ( refs ). for these reasons we also measured duox1 / 2 ( fig1 ), p - erk1 / 2 ( fig1 ), nox3 ( fig1 ) and haras ( fig1 ) protein levels by western blotting in m03 - 13 cells stimulated with igg from control or ms patients in the absence or presence of the ddsk peptide . as shown in the fig1 , 12 , 13 , 14 , igms significantly increased duox1 / 2 , p - erk1 / 2 , nox3 and haras protein levels , and preincubation of cells with ddsk peptide reverted the effect . the ability of ddsk to interfere with 5ht2a receptor downstream signaling measured as duox1 / 2 , p - erk1 / 2 , nox3 and haras protein levels levels , further demonstrate that the peptide is able to revert the biological effects of the autoantibodies present in ms sera on human oligodendrocytes and , therefore , that it may be used for the treatment of multiple sclerosis . on the contrary , incubation of the cells with igg from control subjects decreased duox1 / 2 and p - 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