Patent Application: US-201113581646-A

Abstract:
the present invention relates to a method for the diagnostic and / or prognostic of a neurological disease characterized by an inflammation process in a subject comprising measuring the amount of myeloid derived microvesicles in a cerebrospinal fluid sample obtained from the subject . the invention further relates to a method for predicting and / or monitoring the efficacy of a treatment for a neurological pathology or for monitoring a neurological disease progression .

Description:
to collect csf , rats or mice have been anaesthetized by intraperitoneal injection of 4 % chloral solution and csf has been sampled from the cisterna magna using a glass capillary ( about 10 μl / mouse ; about 100 μl / rat ) and checked for the absence of blood contamination . csf pooled from 2 - 5 rats has been diluted with 0 . 5 - 1 ml ice - cold pbs containing protease inhibitors and subjected to differential centrifugation to obtain three vesicles pellets , p2 , p3 and p4 pellets , as previously described ( bianco et al ., 2009 ). the resulting pellets were either re - suspended in sds sample buffer for western blotting , or re - suspended ( and fixed when needed ) for negative staining electron microscopy or fluorescence microscopy . p2 , p3 and p4 mvs or apoptotic bodies from uv irradiated n9 microglial cells were fixed with 4 % paraphormaldeyde and adsorbed to 400 - mesh formvar / carbon - coated grids ; grids with adherent mvs were then negative contrasted with 1 % uranyl acetate and analysed with philips cm10 transmission electron microscope in bright field . at least 50 vesicles from three different preparations were analyzed from each pellet . immunogold labeling for cd11b / c ( ox42 , harlan sera - lab , uk ) was performed on pelleted mvs , fixed and adsorbed to 400 - mesh formvar / carbon - coated grids , incubated with monoclonal mouse anti - rat ab against cd11b / c for 1 h , followed by 12 - nm gold coupled secondary goat anti - mouse antibody ( jackson immunoresearch laboratories ). mvs were then treated with 1 % gluthaldheyde for 20 ′ and finally subjected to negative staining using 1 % uranyl acetate for 5 min at rt . mvs from primary rat microglia were used as positive control , whereas synaptic vesicles from rat crude synaptome preparation were used as negative controls . mvs pelletted from rat csf at 110 , 000 g for 1 h were re - suspended in about 20 μl of pbs buffer , stained with annexin - v - fitc , cd11b - pe or ib4 - fitc , which all bind extracellular components , spotted on glass microscope slides , covered with glass coverslips , sealed and observed with an inverted zeiss axiovert 200m microscope . staining of fixed mvs with abs directed against intracellular epitopes , i . e . gfap , mbp , iba - 1 and snap - 25 , was performed as follows : primary antibodies was added in a 1 : 1 volume of pbs buffer containing goat serum and 0 . 3 % tritonx - 100 and incubations were allowed for 1 h at rt . primary ab - conjugated mvs were then washed with pbs and pelletted before incubation with fluorochrome - conjugated secondary abs for 2 h at rt and further washing in pbs . re - pelletted labelled vesicles were then spotted on glass microscope slides and observed at fluorescence microscope . csf collected from cx3cr1 - egfp mice was directly stained for cd11b , spotted on glass microscope , sealed and observed with an inverted zeiss axiovert 200m microscope to analyzed the presence of egfp - labelled mvs . p2 , p3 and p4 csf mvs obtained by differential centrifugation from 200 - 500 μl of rat csf were re - suspended in sds sample buffer , loaded on a single lane of a 12 % polyacrylamide gel and blotted onto nitrocellulose filters . 0 . 2 μg of corpus callosum homogenate , 10 μg of rat brain and 1 μg of cortical astrocytes lysate were run in the same gel , as positive controls . selected proteins were detected with specific abs followed by hrp - conjugated secondary abs and revealed using an ecl system . flow cytometry analysis of myeloid csf mvs csf collected from naïve mice or mice exposed to different inflammatory treatments was directly stained with fitc - conjugate isolectin b4 from baindeiraea simplicifolia , ( ib4 - fitc , sigma ), and the phosphatydil serine ligand annexin - v in 1 % bsa . specificity of ib4 labelling was evaluated by treating ib4 - fitc with 1 m melibiose ( 6 - o - α - d - galactopyranosyl - d - glucose , sigma m5500 ) for 30 min before addition to shed mvs ( fig9 ) as previously described ( ayoub a e et al .). csf was then diluted in pbs buffer and labelled mvs were acquired within a fixed time interval on a canto ii hts flow cytometer ( becton dickinson ). data were analysed using fcs 3 software ( becton dickinson , franklin lakes , n . j ., usa ). a gate was established on size using beads of 0 . 5 - 2 μm . whithin this gate ib4 / annexin - v double positive events ( number of events / 0 were evaluated as a parameter of mvs concentration in the csf . samples of human csf collected from lumbar puncture ( 200 - 3000 were stained with ib4 - fitc , annexin v - apc and cd63 - pe . ib4 and annexin - v double positive events were evaluated as a parameter of shed mvs concentration while ib4 and cd63 double positive events was evaluated as a parameter of exosome concentration . the local ethical committee has approved the study and written informed consent from all subjects was obtained . primary cortical astrocytic cultures and purified microglial cultures from e21 embryonic rat pups , were obtained and maintained as previously described ( calegari et al ., 1999 ); ( bianco et al ., 2005a ). murine bv2 microglial cells , originally developed by blasi et al . ( 1988 ) were provided by dr . f . aloisi ( department of cell biology and neuroscience , istituto superiore di sanità , rome , italy ). murine n9 microglia cells , established by prof . castagnoli , were grown as described ( bianco et al ., 2005 ). purified cultures of astrocytes , microglial cells or mixed glial cultures were exposed to lps ( 0 . 4 μg / ml ) or th1 cytokines ( 100 u / ml il1 - β , 200 u / ml tnfα and 500 u / ml ifnγ ) for 24 hours or exposed for different time points ( 24 h , 48 h , 72 h ) to mvs ( p2 and p3 fractions ), pelletted from the supernatants of lps / th1 - primed microglia stimulated with bzatp for 30 min . recipient glial cells were incubated with an amount of shed mvs produced by a double number of microglia ( about 200 mvs / per 25 mm coverslip ). at the end of incubation with different stimuli or mvs , cultured astrocytes were lysed in sds sample buffer for western blotting or harvested with trizol for rt - pcr analysis or fixed with 4 % paraformaldehyde for immunocytochemistry or loaded with the calcium dye fura - 2am for calcium imaging recordings . purified microglia cultures exposed to shed mvs were extracted with trizol for rt - pcr analysis . microglia present in mixed glial cultures , were detached by shaking and analyzed by flow cytometry . in a set of experiments , to evaluate possible internalization of shed mvs into recipient cells , fluorescent shed mvs , released from primary microglia pre - loaded for 1 h with the cytoplasmic fluorescent dye celltracker green cmfda ( 15 μm ), were incubated for 1 h with unstained microglial cells or astrocytes . recipient glial cells were then exposed for 3 min to ib4 - texas red at rt , before being fixed with 4 % paraformaldehyde , to allow the specific staining of mvs attached to the cell surface of recipient cells but not of those internalized into the cell cytoplasm . in addition , mvs isolated from gfp - expressing n9 cells ( bianco et al ., 2005 ), were re - suspended in the culturing medium of control n9 cells and recipient glial cells were fixed 1 hour after shed mvs addition . total rna was isolated from rat primary astrocytes / microglia using mirneasy qiagen kit following the manufacturer &# 39 ; s protocol . to remove any contaminating genomic dna , total rna was digested with dnase . reverse transcriptase was performed using superscript ® iii first - strand synthesis system ( invitrogen ) and oligo ( dt ) 2 o as primer . the resulting cdna was amplified using faststart taq dna polymerase ( roche ) and the following primers for rat pro - inflammatory genes il - 1β ( 339 bp ), sense , 5 ′- cag gaa ggc agt gtc act ca - 3 ′( seq id no . 1 ); antisense , 5 ′- ggg att ttg tcg ttg ctt gt - 3 ′( seq id no . 2 ); il - 6 ( 473 bp ), sense , 5 ′- ccg gag agg aga ctt cac ag - 3 ′( seq id no . 3 ); antisense , 5 ′- tgg tcc tta gcc act cct tc - 3 ′( seq id no . 4 ); inos ( 595 bp ), sense , 5 ′- aag tcc agc cgc acc acc ct - 3 ′( seq id no . 5 ); antisense , 5 ′- tgc aga cgc cat ggt gca gg - 3 ′( seq id no . 6 ); cd206 ( 493 pb ) sense , 5 ′- acc tgg caa gta tcc aca gc - 3 ′( seq id no . 7 ); antisense , 5 ′- ttt tca ggc ctc aat cca ac - 3 ′( seq id no . 8 ); cox - 2 ( 334 ), sense , 5 ′- gag cac ctg cgg ttc gct gt - 3 ′( seq id no . 9 ); antisense , 5 ′- gca gca gcg gat gcc agt ga - 3 ′( seq id no . 10 ). amplified products were electrophoresed on a 2 % agarose gels and visualized by sybr ® safe staining actin ( sense , 5 ′- cta gaa gca ttg cgg tgg acg atg gag gg - 3 ′( seq id no . 11 ); antisense , 5 ′- tga cgg ggt cac cca cac tgt gcc cat cta - 3 ′( seq id no . 12 )) was used to ascertain that an equivalent amount of cdna was synthesized from different samples . cdna synthesis from total rna from rat primary astrocytes / microglia was performed using thermoscript ™ rt - pcr system ( invitrogen ) and random hexamers as primer . il1 - β , il - 6 and inos mrna levels were measured by real time pcr using taqman ® gene expression assays ( rn00580432_m1 *, rn99999011_m1 , rn00561646_m1 *, respectively ) on the abi - prism7500 sequence detection system ( applied biosystems ). the mrna expression was normalized to the label of gapdh mrna ( mm99999915_g1 *). surface stainings for cd11b - pe , cd86 - pe and ib4 - texas red were carried out for 20 min at 4 ° c . or 3 min at rt , before fixing the cells , plated on glass coverslips . iba - 1 , gfap and phalloidin staining was performed on cells fixed with 4 % paraphormaldeyde . nuclei were stained with 4 ′- 6 - diamidino - 2 - phenylindole ( dapi ). cells were mounted and observed with a leica sp5 confocal microscope . after shaking , microglia cells were exposed to anti cd86 - pe antibodies for 30 min a 4 ° c . in the presence of 1 % bsa , washed with pbs , resuspended and analyzed by flow cytometry as described above . at least 5 × 10 4 events / sample were analysed . astrocytes cultured for 2 weeks on 25 - mm - diameter coverslips were exposed to shed mvs for 72 h , loaded with 10 μm fura - 2am for 45 min at 37 ° c . in the culture medium and washed in kreb &# 39 ; s ringer before measuring [ ca 2 + ] i as reported previously ( grumelli et al ., cell calcium ). polychrome iv ( till photonics , grafelfing , germany ) was used as a light source . fura - 2 fluorescence images were collected with a pco super vga sensicam ( axon instruments , forest city , calif ., usa ) and analyzed with tillvision software . after excitation at 340 and 380 nm wavelengths , the emitted light was acquired at 505 nm at 1 - 4 hz . the ratio values in discrete areas of interest were calculated from sequences of images to obtain temporal analyses . calcium concentrations were expressed as f340 / 380 fluorescence ratios . spectrophotometric quantification of nbd - c 6 - hpc - labelled shed mvs released from primary microglia was performed in kreb &# 39 ; s ringer as described previously ( bianco et al ., 2009 ). after overnight pre - treatment with lps ( 0 . 4 μg / ml ) or th1 cytokines ( 100 u / ml il1 - β , 200 u / ml tnfα and 500 u / ml ifnγ ) in the presence or in the absence of minocycline ( 50 μg / ml ), cells were incubated with 50 μm nbd - c 6 - hpc , washed and stimulated with 100 μm bzatp . supernatants were collected , centrifuged 10 min 300 g 4 ° c . to remove cells and debris , and then total fluorescence was assayed at 485 / 535 nm with a spectrophotometric system ( 1420 multilabel counter victor 2 — wallac , finland ). 10 μg of astrocyte lysate were loaded per lane on a 12 % polyacrylamide gel and blotted onto nitrocellulose filters . proteins were detected with specific abs followed by hrp - conjugated secondary abs and revealed using an ecl system . optical density of immuno labelled , bands was measured with image j software vers . 1 . 35i , and average values and se were calculated over three independent experiments . mice ( n = 5 per group ) were anesthetized with 2 , 2 , 2 - tribromoethanol ( 10 mg / ml ; 1 / 27 of body weight ) and the head was placed in a stereotactic injection apparatus ( david kopf instruments , tujunga , calif ., usa ). vesicular stomatitis virus - pseudotyped lentivirus ( lv ) lv - pgk - ifnγ and lv - pgk - tnfα , previously described in muzio et al . ( 2010 ) were injected within the right lateral ventricle at the following coordinates : a . + 0 ; l . + 0 . 8 and d . − 2 . 4 . chronic - progressive eae was induced in c57 / bl6 female mice by immunization with 200 μg / mouse of mog35 - 55 derived peptide as described previously ( furlan et al ., 2009 ). antigens were emulsified 1 : 1 in complete freund adjuvant ( containing ifa plus 4 mg / ml mycobacterium tuberculosis ; strain h37 ) and 300 μl of the final emulsion was administered at the base of the tail ( 100 μl ) and in each flank ( 100 μl per site ) to each mouse . pertussis toxin ( 500 ng / mouse ) was given twice : at the time of the first immunization and 48 hours later . the protocol to induce eae in 6 weeks old female a - smase −/− mice on sv129 background , and their littermates ( kind gift of dr . e schuchman to av ), is identical but with reduced pertussis toxin ( 250 ng / mouse ) in consideration of the young age the authors had to perform immunization to avoid the interference with their inherent neurological phenotype ( horinouchi et al ., 1995 ). to obtain sub - clinical eae for the focal mvs injections , the authors used again the same protocol as for chronic eae , but injecting 50 μg / mouse of mog35 - 55 , and using 250 ng / mouse of pertussis toxin . relapsing - remitting eae was induced in sjl mice by two immunizations , seven days apart , with 150 mg / mouse of plp139 - 151 derived peptide . antigens were emulsified 1 : 1 in complete freund adjuvant ( containing ifa plus 4 mg / ml mycobacterium tuberculosis ; strain h37 ) and 100 μl of the final emulsion were administered in one flank . pertussis toxin ( 500 ng per mouse ) was given twice : at the time of the first immunization and 24 hours later . csf was collected at different time points during the disease , i . e . presymptomatic phase ( 10 dpi , days post injection ), peak ( 20 dpi ) and chronic phase ( 60 dpi ) in chronic - progressive eae model ; pre - clinical phase ( 10 dpi ), first relapse ( 29 dpi ), first remission ( 35 dpi ) and second relapse ( 48 dpi ), in relapsing - remitting eae model . female lewis rats weighing approx . 150gr . were immunized under the skin of the flanks using 1 mg lyophilized spinal cord homogenate emulsified in a total of 200 μl of complete freund adjuvant as described above . body weight and clinical score were recorded daily . clinical scores were assigned according to a standard and validated 0 to 5 scale , described in furlan et al ., 2009 . cumulative disease score was calculated by adding the neurological scores recorded daily for each mouse along the whole period of observation . all efforts were made to minimize animal suffering and to reduce the number of mice used , in accordance with the european communities council directive of nov . 24 , 1986 ( 86 / 609 / eec ). all procedures involving animals were performed according to the guidelines of the institutional animal care and use committee ( iacuc ) of the san raffaele scientific institute . to evaluate formation of focal lesion upon mvs injection , mice with sub - clinical eae ( see above ), 20 days post immunization , were stereotaxically injected in the corpus callosum ( coordinates : 0 mm anterior , 1 . 0 mm lateral to the bregma and 2 . 2 mm in depth ) with mvs ( p2 - p4 fractions ) derived from 24 × 10 6 murine n9 microglial cells dissolved in a total volume of 1 μl of sterile saline . atp , bzatp , lipopolysaccharide ( lps ), mynocycline , fura - 2 / am , fict - and apc - annexin v , ib4 - fitc , ib4 - texas red , melibiose were from sigma - aldrich . celltracker green cmfda , were from molecular probes , pi was bd biosciences from nbd - c 6 - hpc was from invitrogen , il1 - β was from euroclone , tnfα and ifnγ from r & amp ; d . the following antibodies were used : anti mouse cd11b - pe ( bd biosciences ), anti - human cd63 - pe ( bd biosciences ) anti - rat cd86 ( ebioscience ), anti - mouse gfap ( sigma - aldrich ), anti - human calnexin ( sigma - aldrich ), anti - mouse cnpase ( chemicon ), anti - mouse mbp ( chemicon ), anti - mouse snap - 25 smi 81 ( steprnberger monoclonals ) anti - mouse iba - 1 ( wako ). fty720 was purchased from merck chemicals . brain tissue sections were fixed , embedded in paraffin and stained with h & amp ; e , luxol fast blue , and bielshowsky to reveal perivascular inflammatory infiltrates , demyelinated areas and axonal loss , respectively . infiltrating microglia and t cells were stained using ib4 and anti - cd3 and revealed using a biotin - labeled secondary anti - rat antibody . inflammatory infiltrates , demyelinated areas and axonal loss were quantified on an average of 10 complete cross - sections of spinal cord per animal . perivascular inflammatory infiltrates , t cells , and macrophages were evaluated as the number per mm 2 , while demyelinated areas and axonal loss were expressed as the percentage per mm 2 . all data are presented as means or median ± sd , from the indicated number of experiments . data were compared using the student &# 39 ; s t - test for paired or unpaired data or the mann - whitney u - test for non - parametric data . differences were considered to be significant if p & lt ; 0 . 05 and are indicated by an asterisk ; those at p & lt ; 0 . 01 are indicated by double asterisks . the authors enrolled 98 patients and controls whose csf was sampled and analyzed for the presence of ib4 + microvesicles . the authors had 7 controls age and sex matched with patients affected by multiple sclerosis ( mean age 28 years ). these controls were patients undergoing knee surgery under spinal anesthesia but neurologically healthy . the authors collected further 9 sex and age matched controls to the alzheimer patients ( mean age 64 years ) that were either surgery patients undergoing spinal anesthesia , or patients with hydrocephalus , or patients with headache undergoing spinal tap for diagnostic reasons . new neurological patients were divided as follows : 21 ad patients , 5 possible ad patients , 4 patients with frontotemporal dementia ( ftd ), 7 patients with mild cognitive impairment ( mci ), 1 patient with amiloidosis , 15 ms patients , 5 patients with clinically isolated syndrome ( cis ), 2 patients with chronic inflammatory demyelinating polineuropathy ( cidp ), 5 patients with poliradiculitis , 17 patients with other neurological diseases ( ond ). spinal taps were immediately sent to the laboratory . two hundred microliters are used to measure microvesicles concentration as already described by facs . new analysis criteria were set by eliminating non - specific fluorescent signal lining at 45 °. the remaining sample was subjected to differential centrifugation to eliminate cells and cell debris , and finally microvesicles were pelletted at high speed centrifugation and pellet stored in trizol ™ for rna analysis . polychrome v ( till photonics , grafelfing , germany ) was used as a light source . fura - 2 fluorescence images were collected with a pco super vga sensicam ( axon instruments , forest city , calif ., usa ) and analyzed with tillvision software . after excitation at 340 and 380 nm wavelengths , the emitted light was acquired at 505 nm at 1 - 4 hz . the ratio values in discrete areas of interest were calculated from sequences of images to obtain temporal analyses . calcium concentrations were expressed as f340 / 380 fluorescence ratios . living neurons were exposed annexin - v - fitc , fixed with 4 % paraphormaldeyde and counterstained with antibodies direct against the neuronal marker snap - 25 . apoptotic processes , labelled by annexin - v , were quantified by the image j software and normalized to snap - 25 immunoreactivity , as an index of neurite density . alternatively , living cells were exposed to pi , fixed and double stained for α - tubulin and hoescht . pi positive nuclei were quantified and normalized to total hoescht positive nuclei . α - tubulin staining was carried out to evaluate integrity of neuronal cytoskeleton . at least 10 different fields from three independent experiments have been analyzed . the authors found , by negative staining electron microscopy ( em ), membranous mvs , similar in size to both shed microparticles ( mps ) and exosomes released from cultured glial cells , in the csf collected from healthy rodents ( fig1 a ). exosomes typically express tetraspan proteins such as cd63 and cd9 ( cocucci et al . 2009 ), while shed mps are characterized by high levels of phosphatidyl serine ( ps ) ( bianco et al ., 2009 ) on the outer membrane leaflet and bind the ps ligand annexin - v ( fig1 b - d ). by flow cytometry , the authors confirmed the presence of both annexin - v positive shed mps and cd63 positive exosomes ( fig1 e ). the authors excluded the presence of apoptotic bodies , which are much larger and denser than mvs found in the csf ( fig2 a - c ). confocal microscopy and wb analysis of the csf collected from healthy rodents revealed that mvs display either neuronal , astrocytic , oligodendroglial ( fig2 a ), or microglia / macrophage markers ( iba - 1 , ib4 , cd11b ) ( fig1 f - h ), thus indicating they can originate from different brain cells . the microglia / macrophage origin of a mvs subset was definitely confirmed by immunogold em with antibodies to cd11b / c ( ox42 ) ( fig1 i and fig3 a - b ) and by fluorescence microscopy analysis of the csf collected from cx3cr1 - egfp transgenic mice ( jung et al . 2000 ) ( fig1 f ), in which microglia / macrophages express egfp . since blood - born macrophages are virtually absent in the healthy brain , the presence of egfp - labelled mvs in the csf suggests that they derive from resident microglia . thus , the healthy csf drains mvs from all the neural cell lineages tested , including those , like neurons and oligodendrocytes , that are strictly parenchimal and have no contact with liquoral spaces . the authors and others have shown that both the typical danger signal atp and the bacterial membrane component lipopolysaccharide ( lps ) enhance the release of mvs from microglia / macrophages ( bianco et al . 2009 , qu et al ., 2009 ). the authors here show that pre - treatment with the microglia inhibitor mynocicline largely inhibits the increase in shed mps induced by atp in resting and lps - primed primary microglia ( fig1 j ). in addition , the authors show that th1 cytokines ( tnf - α , inf - γ and il1 - β ), which induce formation of blebs at the cell surface characterized by accumulation of iba - 1 ( fig1 k ), are the best priming stimuli to mediate shedding of mps ( fig1 j ). all together , these data indicate that mp shedding takes place very efficiently in microglia exposed in vitro to a pro - inflammatory environment . to verify whether production of both shed mps and exosomes from microglia / macrophages may increase during brain inflammation , the authors stereotaxically injected mice into the ventricular cavity with lentiviral vectors codifying for inf - γ or tnf - α . this treatment induces expansion of activated microglia and recruitment of macrophages from the periphery to periventricular areas of the lateral ventricles and to choroid plexi ; this process typically occurs during experimental autoimmune encephalomyelitis ( eae ), the animal model for multiple sclerosis ( ms , kivisakk et al ., 2009 ) ( fig4 a ). flow cytometry analysis demonstrated that ib4 + microglia / macrophage - derived mvs ( fig5 ) were significantly increased in the csf of mice receiving pro - inflammatory cytokine - expressing vectors ( fig4 b ). microglia / macrophage - derived mvs represented the majority of total csf mvs in tnf - α - ( fig4 c ) or ifn - γ - injected mice ( not shown ), while their percentage was under 50 % in mice injected with the control vector . these results represent the proof of principle that the amount of microglia / macrophage - derived mvs in the csf reflects the activation state of microglia / macrophages in vivo . the pathological hallmark of eae / ms is the presence within the brain of inflammatory infiltrates containing blood derived macrophages and activated microglial cells . consistently , in mice affected by chronic ( c - eae ) or relapsing eae ( r - eae ), mimicking the two most common clinical forms of ms ( hemmer et al ., 2002 ), ib4 + mvs increased significantly , representing over 90 % of total csf mvs ( fig4 c ). the amount of microglia / macrophages mvs was closely associated to the disease course , peaking at onset and during clinical relapses , and decreasing in the chronic phase of the disease ( fig4 e - f ). electron microscopy analysis of the csf of eae rats revealed that the increase of csf mvs during neuroinflammation was not due to the presence of apoptotic bodies , but to the increase in mvs of larger size , likely shed mps ( fig4 g - h ). choroid plexi have been identified as the site of first entry of inflammatory cells during eae ( kivisakk et al ., 2009 ), and , being in direct contact with the csf , may represent the more plausible source of mvs during neuroinflammation . however , the presence in the healthy csf of mvs derived from neurons and oligodendrocytes ( fig2 a ), which are not in direct contact with liquoral spaces , suggests that also mvs released from parenchimal microglia can enter the csf . to define whether microglia / macrophages - derived mvs represent causative and / or amplifying agents of inflammation , primary glial cells were exposed in vitro to mps shed from either th1 / lps - primed or unstimulated microglia and the glial response to mps was analyzed . addition of mps up - regulated astrocytes &# 39 ; mrnas levels of il1 - β , il6 , inos , and cox - 2 ( fig6 d - e ), and induced changes in cell morphology . astrocytes appeared hypertrophic , displayed numerous thicker processes ( fig6 a ), up - regulated the expression levels of gfap ( fig6 b ) and showed increased cytoplasmic calcium concentration ( fig6 c ). primary microglia responded in vitro to shed mps derived from either th1 / lps - primed and un - stimulated microglial cells by up - regulating the t cell co - receptor ligand cd86 on the cell surface ( fig6 h - i ), suppressing the expression of the mannose receptor cd206 , associated with tissue repair ( mantovani et al ., 2004 ; michelucci et al ., 2009 ; pluchino et al ., 2008 ) ( fig6 f ), and increasing the expression of inflammatory markers such as inos , il - 6 and il - 1β ( fig6 f - g ). acquisition of reactive glial phenotype was likely a consequence of internalization of shed mps into recipient cells ( fig7 a - c ). the transfer of mvs between glial cells spontaneously occurred in cultures , as indicated by the presence of gfp + mvs derived from gfp - p2x7 - expressing microglia inside adjacent astrocytes ( fig7 d ). in addition , the authors also found that shed mps can target neighbouring neurons , enhancing glutamatergic transmission ( data not shown ). altogether , these data suggest that shed mps by propagating a reactive phenotype to neighbouring glial cells and affecting neuronal exocytosis , may critically contribute to amplify inflammation in the cns . to evaluate if this occurs also in vivo , the authors stereotaxically injected mvs derived from cultured microglia into the corpus callosum of mice affected by subclinical eae . hystological examination of eae brain revealed numerous perivascular inflammatory foci , close to the site of mvs injection ( fig8 a - b ), which were not present in saline injected controls . these data strongly suggest that mvs play an active role in inducing cns neuroinflammation . the authors have previously shown that mvs shedding is inhibited by acid sphingomyelinase ( a - smase ) inhibitors and is abolished in glial cell cultures established from acid sphingomyelinase ( a - smase ) knock - out mice ( bianco et al ., 2009 . the authors therefore induced eae in a - smase −/− mice and found lower levels of microglia / macrophages mvs in their csf relative to wt mice ( fig9 c ); this was associated to decreased cns infiltration by inflammatory cells ( fig8 c - d ), inhibited disease development ( fig8 e ; fig9 a - b ), and absence of tissue damage ( fig9 d - e ), further suggesting that shed mvs may have effector functions during eae . notably , the immunomodulator drug fty720 , effective in ms treatment , has been recently identified as an inhibitor of a - smase by a mechanism similar to other tricyclic antidepressants and , consistent with our previous finding ( bianco et al ., 2009 ), potently inhibits mp shedding ( fig1 ). to verify if the amount of csf mvs reflects microglia / macrophage activation in humans , the authors collected csf from healthy donors , patients with clinically isolated syndrome ( cis ), considered an initial and acute stage of multiple sclerosis ( ms ), and patients with definite diagnosis of ms . higher levels of microglia / macrophage mvs were detected in the csf from cis and ms patients , as compared to healthy controls ( fig8 e ). notably , mvs isolated from csf of ms patients propagate inflammation ( data not shown ). as compared to young control subjects , patients affected by cis had a significantly higher number of mvs / μl , as did patients with ms although at lower levels ( fig8 f ). it is tempting to speculate that in cis , closer to an acute event , microglia is more activated and therefore levels are higher than in established ms patients , often undergoing lumbar puncture during a stable , remitting phase of the disease . since differences among controls and established ms patients are not easily appreciated on the logaritimic scale in fig8 f , young controls and ms patients are also shown on a linear scale and as bar graph ( fig8 g ). mann - whitney was used to test for statistical significance . to determine whether the findings obtained with the eae rodent models could be extended to human , the authors also collected csf from patients affected by several neurological disorders including ms and alzheimer &# 39 ; s disease ( ad ). the authors found that both total and myeloid mvs were significantly increased , as compared to healthy donors , in ms patients , but also in patients affected by inflammation of the peripheral nervous system ( involving also spinal roots , close to the spinal cord , like guillan - barré , cidp , and radiculitis ) ( fig1 a , b ). furthermore , differently from other neurodegenerative disorders , also ad patients displayed a significant increase in total and myeloid mvs . these results indicate that myeloid mvs are readily detectable and increased in inflammatory and neurodegenerative disorders characterized by microglial activation . as compared to older control subjects , only demented patients from ad group display a dramatically increased number of liquoral mvs , highly significant from a statistical point of view . also in this case this finding is in agreement with knowledge that in ad microglia is widely activated in the brain while in other forms of dementia this phenomenon is limited ( fig1 a ). with the exception of some outliers , most patients with other neurological disorders ( amiloidosi , carcinomatosis , lewis body ) don &# 39 ; t have increased mvs in the csf . only by collecting further samples of the same type , it can be ascertained if the outliers represent true positives in pathologies that have microglia activation ( fig1 b ). on pelletted mvs obtained from the csf of ad and control samples , the authors extracted rna and performed real time rt - pcr for the detection of il - 1β mrna using pre - developed assays by applied biosystems . measures were normalized using 18s rna . surprisingly , ad patients showed a very significant decrease in il - 1β mrna in their mvs , suggesting an anti - inflammatory phenotype of the microglia of origin , as also suggested by other evidence available in literature ( fig1 ). in addition , the present data indicate that mvs shed from primary microglia do not significantly affect neuron viability in vitro , assessed up to 5 days after mv addition to hippocampal neurons ( not shown ). it has been recently shown that lipids such as sphingolipids and gangliosides disassemble amyloid fibrils , which are highly stable and biologically inert structures , into oligomers or other prefibrillar precursors , which are the main neurotoxic amyloid species ( martins et al ., 2008 ). in addition , recent evidence indicated that amyloidogenic polypeptides can interact and penetrate into lipid bilayers of living cells as well as bilayers of artificial vesicles and promote vesicle leakage ( friedman and caflisch , 2009 ). these observations opened the possibility that lipids , present in mv bilayer , can promote neurotoxicity of amyloidogenic peptides , and that amyloid polypeptides can favour mv disruption , inducing the release of neurotoxic compounds , like inflammatory cytokines , stored inside mvs . this hypothesis prompted the authors to investigate the effects of mvs pre - exposed to aβ1 - 42 on cultured neurons ( fig1 ). the authors found that 1 h exposure to mvs pre - incubated overnight with aβ1 - 42 but not aβ1 - 42 or mvs alone strongly affect the number of viable cultured neurons , as indicated by annexin - v staining ( fig1 g ) or propidium iodide ( pi ) uptake ( fig1 c - e ). dendrites of neurons exposed to aβ1 - 42 - treated - mvs have a frequently fragmented or beaded appearance ( fig1 d ), display abnormal cytoplasmic calcium concentration ( fig1 a ) and reduced responsiveness to glutamate agonists ( fig1 b ). interestingly , neuronal damage induced by aβ1 - 42 - treated mvs is strongly inhibited by glutamatergic antagonists ( fig1 f - g ), suggesting an excitotoxicity insult . similar detrimental effects are induced in cultured neurons by mvs isolated from ad csf , pre - incubated with aβ1 - 42 ( fig1 h - i ). finally , primary mouse microglia was pre - treated , or not , with fty720 , also named fingolimod or gylenia ( fig1 ), currently in course of registration for the treatment of multiple sclerosis , for 18 h , then atp was added for 30 min . to induce the release of annexinv + shed vesicles , as measured by facs . treatment with fty720 completely prevented the release of shed vesicles above background levels . since fty720 is known to readily cross the blood brain barrier and to have effect also in the cns of ms patients , these results may contribute to explain its beneficial activity in the cns during neuroinflammation . therefore , the number of shed vesicles in the csf of treated patients represents a biomarker of therapeutic efficacy of ms or other neurological disorders therapeutic treatment . it is then concluded that mvs of different origin can be detected in healthy csf . the csf is recipient of mvs carrying identity markers from all neural lineages , but myeloid mvs represent the major population . in addition , neuroinflammation enhances the release and modifies the content of myeloid mvs in the csf . beside their pathogenic role , mvs produced by microglia leaking into the csf represent a source of qualitative and quantitative information on microglia activation in vivo in humans , thus providing reliable biomarkers to diagnose and / or monitor the pathology course and the effectiveness of drugs targeting activated microglial cells . bianco f et al ., embo j . 2009 28 ( 8 ): 1043 - 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