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Human oligodendroglial cells express low levels of C1 inhibitor and membrane cofactor protein mRNAs Hosokawa, Masato; Klegeris, Andis; McGeer, Patrick L Aug 24, 2004

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ralssBioMed CentJournal of NeuroinflammationOpen AcceResearchHuman oligodendroglial cells express low levels of C1 inhibitor and membrane cofactor protein mRNAsMasato Hosokawa, Andis Klegeris and Patrick L McGeer*Address: Kinsmen Laboratory of Neurological Research, University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC, V6T 1Z3, CanadaEmail: Masato Hosokawa - mhosokaw@interchange.ubc.ca; Andis Klegeris - aklegeri@interchange.ubc.ca; Patrick L McGeer* - mcgeerpl@interchange.ubc.ca* Corresponding author    AbstractBackground: Oligodendrocytes, neurons, astrocytes, microglia, and endothelial cells are capableof synthesizing complement inhibitor proteins. Oligodendrocytes are vulnerable to complementattack, which is particularly observed in multiple sclerosis. This vulnerability may be related to adeficiency in their ability to express complement regulatory proteins.Methods: This study compared the expression level of complement inhibitor mRNAs by humanoligodendrocytes, astrocytes and microglia using semi-quantitative RT-PCR.Results: Semi-quantitative RT-PCR analysis showed that C1 inhibitor (C1-inh) mRNA expressionwas dramatically lower in oligodendroglial cells compared with astrocytes and microglia. ThemRNA expression level of membrane cofactor protein (MCP) by oligodendrocytes was alsosignificantly lower than for other cell types.Conclusion: The lower mRNA expression of C1-inh and MCP by oligodendrocytes couldcontribute to their vulnerability in several neurodegenerative and inflammatory diseases of thecentral nervous system.BackgroundResident brain cells including oligodendrocytes [1,2],astrocytes, astrocytomas, microglia, glioblastomas [3-14],neurons [15,16], neuroblastomas [17,18] and endothelialcells [19,20] express mRNAs for complement proteins.Although the role of complement expression by these cellsremains unclear, local complement activation in the cen-tral nervous system (CNS) might damage these cells andcontribute to the pathology in several inflammatory andneurodegenerative diseases including multiple sclerosis,Alzheimer's disease and progressive supranuclear palsy.For self-protection, resident brain cells also express com-plement inhibitors, such as membrane cofactor protein(MCP), decay-accelerating factor (DAF), CD59, and C1-esterase inhibitor (C1-inh). The human HOG oligoden-droglial cell line produces MCP, DAF, CD59, C1-inh andS-protein, but not complement receptor 1 (CR1) [1].Human oligodendrocytes have been reported to expressCD59 [21] and DAF, but not MCP, CR1, homologousrestriction factor (HRF: C8 bp) or clusterin [22]. Astro-cytes [23], neurons and Schwann cells have been reportedto express CD59 [24] and neuroblastoma cell lines C1-inhPublished: 24 August 2004Journal of Neuroinflammation 2004, 1:17 doi:10.1186/1742-2094-1-17Received: 20 May 2004Accepted: 24 August 2004This article is available from: http://www.jneuroinflammation.com/content/1/1/17© 2004 Hosokawa et al; licensee BioMed Central Ltd. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 9(page number not for citation purposes)[18]. Astrocytoma cell lines have been reported to expressMCP, DAF, and CD59 [25,26].Journal of Neuroinflammation 2004, 1:17 http://www.jneuroinflammation.com/content/1/1/17In this study, the expression level of mRNAs for variouscomplement inhibitors by human oligodendrocytes,astrocytes and microglia were compared by semi-quanti-tative PCR. We show that oligodendrocytes expressextremely low levels of mRNA for C1-inh and significantlylower levels of mRNA for MCP compared with astrocytesand microglia. The expression level of mRNAs for CD59and DAF showed no significant differences between thethree cell types.MethodsCell culture: microglial- and astrocyte-enriched culturesHuman microglial and astrocytic cells were isolated fromsurgically resected temporal lobe tissues. We thank Dr. J.Maguire, Department of Pathology and Laboratory Medi-cine, Vancouver General Hospital for providing the surgi-cal specimens. Isolation protocols described by De Grootet al. [27,28] were used with minor modifications. Tissueswere placed in a sterile Petri dish, rinsed with Hank's bal-anced salt solution, and visible blood vessels wereremoved. After washing tissues two more times withHank's balanced salt solution, tissues were chopped intosmall (<2 mm3) pieces with a sterile scalpel. The frag-ments were transferred into a 50 ml centrifuge tube con-taining 10 ml of 0.25% trypsin solution (Gibco-BRL, LifeTechnologies, Burlington, ON, Canada), and incubated at37°C for 20 min. Subsequently DNase I (from bovinepancreas, Pharmacia Biotech, Baie d'Urfé, PQ, Canada)was added to reach a final concentration of 50 µg/ml. Tis-sues were incubated for an additional 10 min at 37°C.The cell suspension was diluted with 10 ml of Dulbecco'smodified Eagle's medium (DMEM) and nutrient mixtureF12 ham (DMEM-F12; Sigma-Aldrich, Oakville, ON, Can-ada) with 10% fetal bovine serum (FBS; Gibco-BRL, LifeTechnologies), and gently triturated by using a 10 mlpipette with a wide mouth. After centrifugation at 275 × gfor 10 min, the cell pellet was resuspended in serum con-taining medium, triturated several times, and passedthrough a 100 µm nylon cell strainer (Becton Dickinson,Franklin Lakes, NJ). The cell suspension was then centri-fuged once more (275 × g for 10 min), resuspended into10 ml of DMEM-F12 with 10% FBS containing gen-tamicin (50 µg/ml, from Sigma), and plated ontouncoated 10 cm tissue culture plates (Becton Dickinson).Plates were placed in a humidified 5% CO2, 95% airatmosphere at 37°C for 2 hr in order to achieve adherenceof microglial cells. Non-adherent cells with myelin debriswere removed from these microglia-enriched cultures andtransferred into poly-L-lysine coated 10 cm tissue cultureplates in order to achieve adherence of astrocytes. Plateswere incubated for 48 hr, after which the culture mediumcontaining myelin debris and non-adherent cells wasremoved and used to prepare oligodendroglial cell cul-mRNAs were extracted. Immunostaining with antibodiesagainst CD68 (Dako, Mississauga, ON, Canada) whichstains microglia as well as macrophages, and glial fibril-lary acidic protein (GFAP, Dako), which is a marker ofastrocytes, showed that the microglia-enriched culturescontained 93.5 ± 3.6 % (N = 4) microglial cells, whileastrocyte-enriched cultures contained 85.7 ± 3.4 % (N =4) astrocytes.Cell culture: oligodendroglial cellsThese were prepared as described before [2]. Briefly, cellculture media containing myelin debris and non-adherentcells that were removed from astrocyte-enriched cultureswere used to extract oligodendroglial cells. The non-adherent cells were collected by centrifugation at 275 × gfor 10 min and replated onto uncoated 10 cm tissue cul-ture plates for another 24 hr. Subsequently, the cell cul-ture medium containing floating cells was transferred to50 ml tubes and Lymphoprep solution (Axis-Shield, Oslo,Norway) used to reduce the amount of contaminatingmyelin debris. For this purpose, 10 ml of Lymphoprepsolution was carefully placed under the oligodendrocytecell suspension and the density gradient was centrifugedat 325 × g for 10 min. The interphase was collected andtransferred to a 50 ml centrifuge tube. Fresh culturemedium was added and the suspension was centrifuged at275 × g for 7 min. The cell pellet was resuspended and theoligodendrocyte cultures seeded onto 60 mm plastic cul-ture dishes. Immunostaining with anti-O4 antibody(Chemicon International, Temecula, CA), which is amarker of oligodendrocytes, showed that the oli-godendrocytes-enriched cultures contained 95.3 ± 4.4 %(N = 4) oligodendrocytes.RNA isolation and cDNA synthesis by reverse transcriptionTotal RNA from oligodendroglial cells, microglia, andastrocytes were isolated by the acid guanidium thiocy-anate-phenol-chloroform method. Two µg of the RNAwas then used to prepare cDNA. RNA was treated with 10U of DNase I (Gibco BRL, Life Technologies) for 60 minat 37°C in 25 µl of 1 × reverse transcriptase buffer (50 mMTris-HCl, 75 mM KCl, 3 mM MgCl2) containing 40 U ofRNase inhibitor (Pharmacia Biotech) and 1 mM dithioth-reitol (DTT), following by incubation at 85°C for 5 minto inactivate the enzyme. Reverse transcription was per-formed at 42°C for 90 min in 50 µl of the following mix-ture: 1 × reverse transcriptase buffer containing 2 µg ofRNA, 5 mM DTT, 0.2 µg random hexamer primers (Phar-macia Biotech), 1 mM deoxynucleotides (Gibco BRL, LifeTechnologies), 40 units of RNase inhibitor, and 400 unitsof SuperScript II reverse transcriptase (Invitrogen LifeTechnologies, Burlington, ON, Canada). At the end of theincubation period, the enzyme was inactivated by heatingPage 2 of 9(page number not for citation purposes)tures as described below. Both microglial- and astrocyte-enriched cultures were grown for 6 to 7 days before theirat 65°C for 10 min [29].Journal of Neuroinflammation 2004, 1:17 http://www.jneuroinflammation.com/content/1/1/17Polymerase chain reactionPCR amplification was carried out in a 25 µl reaction mix-ture containing 1 × GeneAmp PCR buffer II (Perkin Elmer,Foster City, CA), 1.25 units AmpliTaq Gold DNApolymerase (Perkin Elmer), 2 mM MgCl2 (Perkin Elmer),200 µM dNTPs (Gibco BRL, Life Technologies) and 0.5µM of each specific primer (Table 1). The mixture was pre-pared before the addition of 1.25 µl of cDNA. PCR ampli-fication was carried out using an MJResearch (Boston,MA) programmable thermal controller. The amplificationprogram consisted of an initial denaturation step at 94°C,which was extended to 9 min in order to activate Ampli-Taq Gold enzyme. The remaining cycles were 1 min at94°C, 1 min at 55°C and 1 min at 72°C. The number ofcycles performed was 27 for glyceraldehyde-3-phosphatedehydrogenase (G3PDH), 30 for CD59, C1-inh and MCP,and 32 for DAF. After amplification, PCR products wereseparated on a 6% polyacrylamide gel and visualized byincubation for 10 min in a solution containing 10 ng/mlof ethidium bromide. Polaroid photographs of the gelswere taken.PCR primer design and restriction analysesPrimers were designed to span introns so that cDNA-derived PCR products would be of different sizes to thoseproduced if genomic DNA was amplified (see Table 1).DAF and MCP were exceptions, since only cDNAsequences were available. Primers were synthesized eitherby Sigma-Aldrich or ID Labs (London, ON, Canada). Theprimer sequences and predicted PCR fragment sizes arelisted in Table 1, along with the names of the enzymesused for restriction digest analysis of each PCR fragment.The restriction digestion reactions were carried out at37°C for 2 hr in the presence of 1 × the appropriate bufferprovided by the suppliers (Invitrogen, Life Technologiesand New England Biolabs, Mississauga, ON, Canada). Thedigested PCR products were analyzed on a 6% polyacryla-Statistical analysisThe data are presented as means ± s.e.m. The significanceof difference between values was estimated by means ofone-way analysis of variance (ANOVA) with Fisher's LSDpost-hoc test. P < 0.05 was considered to show statisticallysignificant differences.Double fluorescence immunocytochemical analysisOligodendrocytes, astrocytes, and microglia were har-vested and air-dried on glass slides. Cells were then fixedwith 4% paraformaldehyde for 10 min and permeabilizedwith 0.2% Triton X-100 in phosphate-buffered saline(PBS) for 5 min. For inactivation of endogenous peroxi-dase, cells were incubated with 0.3% H2O2 for 30 min.Blocking was performed for 1 hr at room temperature in5% skim milk.For double fluorescence immunostaining, cells were incu-bated at room temperature overnight with a primary anti-body in 1% normal serum. The primary antibody and thedilution used in the first cycle were as follows: O4(Chemicon International, 1: 100) for oligodendrocytes,GFAP (Dako, 1: 10,000) for astrocytes, CD68 (DAKO, 1:50) for microglia. Cells were then treated for 2 hr at roomtemperature with a biotin conjugated anti-mouse IgM(Vector Laboratories, Burlingame, CA, 1: 200) secondaryantibody for O4, a biotin conjugated anti-rabbit IgG (Vec-tor Laboratories, 1: 200) secondary antibody for GFAPand a biotin conjugated anti-mouse IgG (Vector Laborato-ries, 1: 200) secondary antibody for CD68. Then cellswere incubated with Texas Red Avidin DCS (Vector Labo-ratories) for 1 hr. The primary antibody and the dilutionused in the second cycle were as follows: for C1-inh, goatanti-C1-inhbitor (Quidel, San Diego, CA, 1: 50); forCD59, mouse anti-CD59 (Serotec Ltd, Oxford, UK, 1: 10)or rat anti-CD59 (Serotec, 1: 25). Cells were incubated at4°C for 3 days with a primary antibody in 1% serum cor-Table 1: Oligonucleotide primers used for PCR, and the corresponding restriction endonucleases used for product confirmation.Gene Sequence (5' → 3') Fragment size (introns)Genbank accession NoRestriction enzymes used and the expected sizes of digestion products (bp)C1 inh-F GTT GGG GGA TGC TTT GGT AGA TTT C 332 M13690 Sau 3AI (246, 86)C1 inh-R TTA GGA CTC TGG GGC TGC TGC TGT A (2 introns)CD59-F CTG CTG CTC GTC CTG GCT GTC TTC T 280 M34671 Pst I (233, 47)CD59-R TCC CAC CAT TTT CAA GCT GTT CGT T (2 introns)MCP-F CAA TTC AGT GTG GAG TCG TGC TGC 265 Y00651 Sau 3AI (193, 72)MCP-R TGA GGC ACT GGA CGC TGG AGA T (unknown)DAF-F GTA CTG TGA ATA ATG ATG AAG GAG 364 M30142 Hae III (330, 34)DAF-R TCT TAA CTC TTC TTT GGC TAA GTC (unknown)G3PDH-F CCA TGT TCG TCA TGG GTG TGA ACC A 251 X01677 Dde I (168, 83)G3PDH-R GCC AGT AGA GGC AGG GAT GAT GTT C (2 introns)Page 3 of 9(page number not for citation purposes)mide gel (data not shown). In all cases the restriction frag-ments observed were of the predicted size (see Table 1).responding to the secondary antibody type. Cells werethen treated for 2 hr at room temperature with FITC-con-Journal of Neuroinflammation 2004, 1:17 http://www.jneuroinflammation.com/content/1/1/17jugated anti-mouse IgG (Vector Laboratories, 1: 200),anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA,1: 200), or anti-rat IgG (Cappel, Durham, NC, 1: 200).The glass slides were then rinsed with distilled water, anda drop of Vectashield mounting medium (Vector Labora-tories) placed on the slide.ResultsRT-PCRRT-PCR was carried out using primers for C1-inh, CD59,DAF and MCP. The housekeeping gene G3PDH wasamplified in parallel with each RT-PCR run as an internalstandard. Figure 1 illustrates the bands obtained for eachof the RT-PCR products from oligodendrocytes (Fig. 1A),astrocytes (Fig. 1B) and microglia (Fig. 1C). Specificity ofeach of the products was established by endonucleasedigestion (Table 1).Semi-quantitative RT-PCR analysisTo compare the ratio of each of the complement inhibi-tors to G3PDH, statistical analysis was performed bymeans of one-way ANOVA with Fisher's LSD post-hoc test(Fig. 2). The overall mean ± s.e.m. for C1-inh/G3PDH was0.55 ± 0.12 (N = 5) in astrocytes, 0.58 ± 0.09 (N = 3) inmicroglia and 0.09 ± 0.06 (N = 12) in oligodendrocytes(Fig. 2A). Oligodendrocytes showed a highly significantdifference from astrocytes and microglia (Fig. 2A; P <0.001 by one-way ANOVA with Fisher's LSD post-hoctest). For MCP/G3PDH, the ratios were 0.80 ± 0.22 (N =5) in astrocytes, 0.93 ± 0.10 (N = 3) in microglia and 0.44± 0.19 (N = 12) in oligodendrocytes. Oligodendrocytesshowed a significant difference from astrocytes and micro-glia (Fig. 2B; P = 0.002 vs. astrocytes and P = 0.001 vs.microglia by one-way ANOVA with Fisher's LSD post-hoctest). The corresponding means for CD59/G3PDH were0.73 ± 0.10 (N = 5) in astrocytes, 0.83 ± 0.03 (N = 3) inmicroglia and 0.76 ± 0.09 (N = 14) in oligodendrocytes(Fig. 2C). The corresponding means for DAF/G3PDHwere 0.67 ± 0.07 (N = 5) in astrocytes, 0.67 ± 0.07 (N = 3)in microglia and 0.66 ± 0.15 (N = 14) in oligodendrocytes(Fig. 2D). There were no significant differences betweenthe three cell types for CD59 and DAF. Each N representsa different patient.Double fluorescence immunohistochemistryIn order to establish identity between oligodendroglialcells, astrocytes or microglia and cells expressing the com-plement inhibitor proteins CD59 or C1-inh, double fluo-rescence immunostaining was carried out.Oligodendrocytes were detected by O4 staining with aTexas Red tagged secondary antibody (Fig. 3A and 3D) inthe first cycle and CD59 (Fig 3B) or C1-inh staining (Fig.3E) detected with a green FITC tagged antibody in the sec-the first cycle and CD59 staining (Fig 3H) or C1-inh stain-ing (Fig. 3K) detected with a green FITC tagged antibodyin the second cycle. Microglia were detected by CD68staining with a Texas Red tagged secondary antibody (Fig.3M and 3P) in the first cycle, and CD59 staining (Fig 3N)or C1-inh staining (Fig. 3Q) detected with a green FITCtagged antibody in the second cycle. With double fluores-cent excitation, all cells fluoresced yellow (Fig.3C,3F,3I,3L,3O,3R), indicating colocalization of O4 withCD59 or C1-inh, GFAP with CD59 or C1-inh, and CD68with CD59 or C1-inh.DiscussionThis report shows that human oligodendrocytes express amuch lower level of mRNA for C1-inh than astrocytes andmicroglia, and a significantly lower level of mRNA forMCP. The mRNA levels of CD59 and DAF were compara-ble in all the three cell types. Overall our data suggest thatoligodendroglial cells, in common with other cell types,can produce complement inhibitors, but at a significantlylower level for C1-inh and MCP.It has already been reported that human neurons andSchwann cells [24], neuroblastoma cell lines [18], astro-cytes [23], astrocytoma cell lines [25,26], the HOGhuman oligodendroglial cell line [1] and oligodendro-cytes [21,22] produce some or all of the complementinhibitor proteins and their mRNAs.Activation of the complement cascade and deposition ofactivated complement fragments occur in non-infectiousdiseases such as multiple sclerosis, Pick's disease, Alzhe-imer's disease and other neurodegenerative conditions[15,16,30-34]. Complement inhibitors may play animportant role in preventing such pathology.Full activation of the complement cascade requires over-coming a series of endogenous inhibitory factors. Oli-godendrocytes are vulnerable to complement attack,which is particularly observed in multiple sclerosis [35-37] and this vulnerability may be related to a deficiency oftheir ability to express complement regulatory proteins[22], particularly C1-inh.Sporadic complement attack, in the form of complementactivated oligodendroglia (CAO) is also observed in anumber of neurodegenerative conditions [38,39], includ-ing Alzheimer's, Pick's, Huntington's and Parkinson's dis-eases, amyotrophic lateral sclerosis, progressivesupranuclear palsy, Shy-Drager syndrome, argyrophilicgrain dementia and pallido-nigral luysial atrophy [38,39].The source of the complement proteins that become acti-vated is unknown, but the data presented here suggest thatPage 4 of 9(page number not for citation purposes)ond cycle. Astrocytes were detected by GFAP staining witha Texas Red tagged secondary antibody (Fig. 3G and 3J) inoligodendrocytes are vulnerable to complement attackbecause of a low expression of C1-inh and MCP.Journal of Neuroinflammation 2004, 1:17 http://www.jneuroinflammation.com/content/1/1/17Demonstration of RT-PCR productsFigure 1Demonstration of RT-PCR products. Polaroid photographs of typical ethidium bromide-stained gels of RT-PCR products from oligodendrocytic (Fig. 1A), astrocytic (Fig. 1B) and microglial (Fig. 1C) RNA extracts. Lanes for individual mRNA products are indicated in the legend at the top. Size markers are in the right lanes. MCP, membrane cofactor protein (265 bp); DAF, decay-accelerating factor (364 bp); CD59 (280 bp); C1-inh, C1-esterase inhibitor (332 bp); G3PDH, glyceraldehyde-3-phosphate dehydrogenase (251 bp).MicrogliaCAstrocytesOligodendrocytesBAMCPDAFCD59C1-inhG3PDHMarker517506396344298220201517506396344298220201517506396344298220201Page 5 of 9(page number not for citation purposes)Journal of Neuroinflammation 2004, 1:17 http://www.jneuroinflammation.com/content/1/1/17A comparison of relative complement inhibitor expression level between oligodendrocytes, astrocytes and microgliaFigure 2A comparison of relative complement inhibitor expression level between oligodendrocytes, astrocytes and microglia. The data were estimated by one-way analysis of variance (ANOVA) with Fisher's LSD post-hoc test (A and B; P < 0.05 was considered Page 6 of 9(page number not for citation purposes)to show statistically significant differences).Journal of Neuroinflammation 2004, 1:17 http://www.jneuroinflammation.com/content/1/1/17Double fluorescence immunohistochemistry of oligodendrocytes, astrocytes and microgliaFig r 3Double fluorescence immunohistochemistry of oligodendrocytes, astrocytes and microglia. Double fluorescence immunostain-ing for O4 and CD59 or C1-inh is demonstrated in A-F. In A and D, cells of typical oligodendroglial morphology were stained in the initial cycle for the specific oligodendroglial marker O4. Detection is by a Texas Red-conjugated secondary antibody. Second cycle staining for CD59 (B) and C1-inh (E) are shown. The detections are by an FITC-linked green fluorescent second-ary antibody. In C and F, double immunofluorescences are shown in which the cells appear yellow, demonstrating colocaliza-tion of O4 with CD59 or C1-inh. Double fluorescence immunostaining of astrocytes for GFAP and CD59 or C1-inh is demonstrated in G-L. In G and J, cells of typical astrocytic morphology are stained in the initial cycle for the specific astroglial marker GFAP. Detection is by a Texas Red-conjugated secondary antibody. Second cycle staining for CD59 (H) and C1-inh (K) is shown with an FITC-linked green fluorescent secondary antibody. In I and L, double immunofluorescences are shown in which the cells appear yellow, demonstrating colocalization of GFAP with CD59 or C1-inh. Double fluorescence immunostain-ing for microglia using the specific marker CD68 and CD59 or C1-inh is demonstrated in M-R. In M and P, cells of typical microglial morphology are stained by CD68 with detection by a Texas Red-conjugated secondary antibody. Second cycle stain-ing for CD59 (N) and C1-inh (Q) are shown. The detections are by an FITC-linked green fluorescent secondary antibody. In O and R, double immunofluorescences are shown in which the cells appear yellow, demonstrating colocalization of CD68 with Page 7 of 9(page number not for citation purposes)CD59 or C1-inh. (Magnification: × 200)Journal of Neuroinflammation 2004, 1:17 http://www.jneuroinflammation.com/content/1/1/17ConclusionsThese results suggest that the lower expression of C1-inhand MCP by oligodendrocytes could contribute to theirvulnerability in several neurodegenerative and inflamma-tory diseases of the central nervous system, particularlymultiple sclerosis.List of abbreviationsanalysis of variance (ANOVA)central nervous system (CNS)complement activated oligodendroglia (CAO)complement receptor 1 (CR1)decay-accelerating factor (DAF)dithiothreitol (DTT)fluorescein isothiocyanate isomer (FITC)glyceraldehyde-3-phosphate dehydrogenase (G3PDH)glial fibrillary acidic protein (GFAP)homologous restriction factor (HRF)membrane cofactor protein (MCP)phosphate-buffered saline (PBS)Competing interestsNone declared.Authors' contributionsMH was responsible for the majority of the experimentalstudies, and for writing the manuscript. 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