Inhibitory effects of microinjection

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CCL2 recruitment of IL-6-producing CD11b+ monocytes

CCL2recruitment of IL-6-producing CD11b +monocytes to the draining lymph nodes during the initiation of Th17-dependent B cell-mediated autoimmunityYing Bai*1,2,6,Ruolan Liu*2,DeRen Huang 3,Antonio La Cava 4,Yi-yuan Tang 1,Yoichiro Iwakura 5,Denise I.Campagnolo 2,Timothy L.Vollmer 2,Richard M.Ransohoff 3and Fu-Dong Shi 1,21Institute of Neuroinformatics and Laboratory for Brain and Mind,Dalian University of Technology,Dalian,China2Barrow Neurological Institute,St.Joseph's Hospital and Medical Center,Phoenix,AZ,USA 3Neuroinflammation Research Center,Department of Neurosciences,Lerner Research Institute,Cleveland Clinic Foundation,Cleveland,OH,USA4Division of Rheumatology,David Geffen School of Medicine,University of California,Los Angeles,CA,USA5Center for Experimental Medicine,Institute of Medical Science,University of Tokyo,Tokyo,Japan6Division of Neurology,Xin Hua Hospital,Dalian University,Dalian,ChinaThe development and function of Th17cells are influenced in part by the cytokines TGF-b ,IL-23and IL-6,but the mechanisms that govern recruitment and activity of Th17cells during initiation of autoimmunity remain poorly defined.We show here that the development of autoreactive Th17cells in secondary lymphoid organs in experimental autoimmune myasthenia gravis –an animal model of human myasthenia gravis –is modulated by IL-6-producing CD11b +cells via the CC chemokine ligand 2(CCL2).Notably,acetylcholine receptor (AChR)-reactive Th17cells provide help for the B cells to produce anti-AChR antibodies,which are responsible for the impairment of the neuromuscular transmission that contributes to the clinical manifestations of autoimmunity,as indicated by a lack of disease induction in IL-17-deficient mice.Thus,Th17cells can promote humoral autoimmunity via a novel mechanism that involves CCL2.Key words:Autoantibodies ÁCCL2ÁMyasthenia gravis ÁTh17Supporting Information available onlineIntroductionHelper CD4+T cells that are characterized by the production of IL-17are commonly called Th17cells [1–6].Th17cells have features that are distinct from those of both Th1and Th2lineages.For example,TGF-b and IL-6are required for the generation ofTh17cells,and IL-23supports the survival and expansion of these cells [1–8].Recent data also suggest that IL-6and TGF-b drive initial lineage commitment of Th17cells,whereas IL-23mediates the full acquisition of pathogenic function of Th17cells [9].Studies in several experimental models have indicated that IL-17is a major mediator of tissue inflammation and autoimmunedisease*These authors contributed equally to this work.Correspondence:Dr.Fu-Dong Shi e-mail:Fu-Dong.Shi@[4–6],but the cellular source of the cytokines and chemokines required for Th17development and recruitment during initiation of autoimmunity have not been investigated as yet.The CC chemokine ligand2(CCL2),initially known as monocyte chemoattractant protein1(MCP-1),is expressed by inflammatory cells at sites of tissue injury[10–13].CCL2directs the migration of monocytes,dendritic cells,NK cells and T cells, and coordinates inflammatory responses during infection[13]. Here we tested the possibility that CCL2-mediated homing of monocytes may affect Th17cells in a mouse model of human myasthenia gravis(MG),the experimental autoimmune MG (EAMG)model.In MG and EAMG,the cardinal sign of weakness is mediated by circulating IgG autoantibodies to the acetylcholine receptor(AChR)or to other muscular antigens at the neuromus-cular junction[14,15].We show here that CCL2plays a critical role in coordinating cognate interactions among IL-6-producing CD11b+cells,auto-reactive Th17cells,and B cells.These interactions appear critical in the genesis of autoantibodies and in subsequent development of EAMG.ResultsExpression of CCL2in EAMGIn C57BL/6(B6)mice,EAMG presents as a protracted,progressive form of muscular weakness[16].This presentation differs from the bi-phasic form of this disease characterized by early muscular inflammatory infiltration in Lewis rats[17].Since the expression of CCL2can be important for the pathogenesis of EAMG in the Lewis rat model[18],we measured CCL2levels in EAMG B6mice at the sites of injections as well as in secondary lymphoid organs (lymph nodes and spleen).By ELISA,CCL2was hardly detectable in naïve mice(0.03Æ0.02pg/mg tissue).However,CCL2 expression was elevated in AChR/CFA-injected mice at the sites of immunization,in lymph nodes,and in spleens(Fig.1).CCL2-deficient mice developed mild muscular weakness upon induction of EAMGTo investigate directly whether CCL2contributes to the develop-ment of EAMG,we immunized CCL2–/–mice and control B6mice with AChR in CFA.Muscular weakness characteristic of MG was apparent in control mice around day14post immunization(p.i.), and became severe after booster immunizations on days30and 60,when the clinical score maximized at2.5Æ0.3.By contrast, no signs of muscular weakness were observed until day25p.i.in the CCL2–/–mice,and their disease remained mild(maximal clinical score of0.6Æ0.2,p<0.01)even after booster immuniza-tions(Fig.2A).Also,the CCL2–/–mice had less muscle AChR loss compared to control mice(Fig.2B).Thus,CCL2–/–mice are developed milder behavioral and histological disease after the induction of EAMG.B cell and autoantibody responses in CCL2–/–miceIn MG and EAMG,major immune effectors of the disease process at the neuromuscular junctions are circulating IgG autoantibodies reactive to AChR,whereas the cellular inflammatory infiltrates in the muscles are sparse and not considered essential for the impaired neuromuscular transmission,both in humans and mice [16,19,20].Therefore,we measured anti-murine AChR IgG(and related isotypes)in CCL2–/–mice and control miceimmunized L2expression in EAMG.C57BL/6(B6)mice were injectedwith AChR in CFA and sacrificed3or7days later.Tissues from theinjection sites as well as from spleens and lymph nodes were preparedas homogenates.The content of CCL2in the supernatant wasquantified by ELISA(n=4mice/group.*p<0.05).L2–/–mice are less susceptible to the induction of EAMG.B6mice and CCL2–/–mice were immunized with AChR/CFA on days0,30and60to induce EAMG.(A)Animals were then evaluated every2–3daysfor characteristic muscle weakness as described in Materials andmethods.(B)Loss of muscle AChR content.The muscle tissues wereremoved from the mice in(A)at the termination of experiments and thepercentage loss of muscle AChR was quantified.All results areexpressed as mean values+SD.**p<0.01.with pared with controls,CCL2–/–mice had only a marginal reduction of total anti-AChR IgG levels (p <0.06)(Fig.3A).Although the levels of anti-AChR IgG1and IgG2a were similar in the two groups,the amounts of anti-AChR IgG2b were markedly lower in the CCL2–/–mice (p <0.01)(Fig.3A).The same results were obtained for anti-torpedo AChR IgG in AChR-immunized control and CCL2–/–mice (control vs .CCL2–/–mice:IgG, 1.6Æ0.5vs .0.7Æ0.2,p =0.42;IgG2b, 1.2Æ0.2vs .0.3Æ0.1,p =0.008).CCL2exerts its function through its receptor CCR2in vivo [11,13].CCR2expression has been recently described on B cells and influences B cell functions [21,22].To determine whether the altered autoantibody response in CCL2–/–mice could be linked to CCR2expression on B cells,we purified B cells from control mice and CCL2–/–mice for studies of proliferation and production of antibodies.As shown in Fig.3B,B cells from control mice and from CCL2–/–mice proliferated at a similar extent after BCR and CD40cross-linking.The number of plasma cells generated from control and CCL2–/–B cells after BCR stimuli were similar (Fig.3C),and so were the levels of total IgG and IgG2b released from plasma cells (Fig.3D).Thus,CCL2deficiency does not impair the capacity of B cells to proliferate,differentiate into plasma cells,or release IgG antibodies.Although the intrinsic features of antibody responses are preserved in CCL2–/–mice,it is possible that CCL2–/–B cells may have an altered capacity in migration during the immune response,since B cells express CCR2.To examine this possibility,we quantified CD19+B cells in several peripherallymphoid organs (spleen and lymph nodes)during the course of EAMG.There was no significant alteration in B cell percentage and numbers between control mice and CCL2–/–mice (Supporting Information Fig.1).It was thus unlikely that the observed phenotype of EAMG in CCL2–/–mice was due to the defects in B cell migration.Impaired Th17responses in CCL2–/–miceThe altered levels of IgG autoantibodies in the AChR-primed CCL2–/–mice,associated with an intact B cell proliferation and differentiation,led us to investigate the phenotype of T cells in this model.In the absence of CCL2,T cell proliferation was not significantly altered,as reflected by CFSE dilution experiments (Supporting Information Fig.2).Nonetheless,the preferential reduction of the levels of anti-AChR IgG2b suggested altered Th cell L2plays a role in the development of both Th2and Th1cells [11,23].In our model,IFN-c production by CD4+T and CD8+T cells in response to AChR stimulation was marginally impaired in CCL2–/–mice (Fig.4A and B;Supporting Information Fig.3),and both CCL2–/–mice and controls produced similar amounts of IL-4(Fig.4D and E).Since the cytokine IL-17is an important mediator of tissue inflammation and Th17cells are important in the pathogenesis of several autoimmune conditions [1–6],we tested the role oftheseFigure 3.B cells are functionally intact in the CCL2–/–L2–/–mice and control mice were immunized with AChR/CFA as described in Materials and methods .(A)Serum samples were collected from mice as decribed in Fig.2at the termination of the experiments.Anti-AChR IgG antibodies and IgG isotypes were determined by ELISA (n =16–18)on AChR-coated plates.Mice were sacrificed on day 14p.i.,and lymph nodes cells were cultured with or without B-cell stimuli.(B)Proliferation of B cells measured as [3H]thymidine incorporation.(C)Plasma cell differentiation after polyclonal B cell stimulation measured by FACS.(D)Antibody production in the culture supernatants of plasma cells from spleen (left bars)and lymph nodes (right bars)(n =4/group).All results are expressed as mean values ÆSD.**p <0.01.cells in EAMG,because it is not known how and/or whether Th17cells contribute to this disease.We first compared Th17cell responses to AChR between wild-type (B6)and CCL2–/–T cells.Interestingly,CCL2–/–T cells failed to mount an effective Th17response to AChR (Fig.4A and C).To determine whether CCL2–/–T cells have intrinsic defects in Th17differentiation,we sorted CD4+CD44+T cells from control mice and CCL2–/–mice and cultured them in the presence or absence of IL-6and TGF-b for 48h.Levels of IL-17were similar for control T cells and CCL2–/–T cells (8834Æ322vs .8923Æ202ng/mL),showing that CCL2–/–Tcells do not have an intrinsic defect in Th17differentiation.The failure in mounting Th17cells in CCL2–/–mice must thus derive from other factors.CCL2modulates the homing of CD11b +cells to lymphoid organsPhenotypic commitment of Th17cells is dependent on IL-6[1–6].To study the mechanism underlying the impaired generation of Th17cells in AChR-immunized CCL2–/–mice,we quantified IL-6in several cell types of EAMG mice.As shown in Fig.5A,CD11b +cells and,to a lesser degree,CD11c +cells were the predominant sources of IL-6.The production of IL-6in the supernatants from cultured spleen and lymph node cells of CCL-2–/–mice was significantly lower than that of controls (Fig.5B).Since CCL2–/–mice had low levels of IL-6,a cytokine mainly produced by CD11b +cells,we sought to address whether CCL2deficiency altered the homing of CD11b +cells to lymph nodes or spleens,i.e .,causing a secondary reduction of IL-6production.We found no significant difference in the numbers and percentages of CD4+,CD8+,NK1.1+CD3–(NK cells)or NK1.1+CD3+(NKT)cells in lymph nodes of control mice and CCL2–/–mice (Supporting Information Fig.4).However,the numbers of CD11b +and CD11b +CD11c +cell populations were reduced in spleens (Fig.5C)and the lymph nodes of CCL2–/–mice (Supporting Information Fig.5).These data suggested that CCL2deficiency may interfere with homing of CD11b +cells and CD11b +CD11c +cells to peripheral lymphoid organs,which in turn reduces IL-6avail-ability for development of Th17cells.IL-6–/–mice fail to mount AChR-induced Th17responsesIn a previous study,IL-6–/–mice were found resistant to the induction of EAMG [24].Our current data taken in the content of recent findings [9]suggest that the resistance to EAMG could derive from faulty development of Th17cells.Since this could be secondary to IL-6deficiency,we measured autoantigen-induced Th17responses in IL-6–/–mice immunized with AChR/CFA.Consistent with previous reports [24],IL-6–/–mice wereresistantFigure 4.Th17responses are impaired in the CCL2–/–mice.Splenocytes from B6and CCL2–/–mice immunized with AChR/CFA were harvested at day 7–14p.i.and stimulated with10l g/mL AChR in culture medium for 3–5days.For intracellular cytokine staining,cells were restimulated with PMA/ionomycin/brefeldin A for further 5h,then IFN-c -,IL-4-,and IL-17-producing cells were detected by flow cytometry as described in Materials and methods .Results were shown in representative plots from three separate experiments (n =4–6mice/group)and bar graphs for summary data.(A–C)AChR-induced IFN-c and IL-17production by CD4+cells.(D,E)AChR-induced IL-4production by CD4+cells.**p <0.01.to the induction of EAMG.Importantly,IL-6–/–mice had impaired capacity to mount AChR-induced production of IL-17in CD4+cells,while IFN-c production was not altered significantly (Fig.6).These results indicate a role of IL-6in the development of Th17cells and not Th1responses in EAMG.IL-17facilitates autoantibody responses and clinical EAMG independently of IFN-cNext,we injected recombinant IL-17into AChR-primed mice during the T cell priming period,and then compared the outcome to that of mice given only PBS injections.Significantly,recipients of IL-17developed severe EAMG and elevated titers of AChR-specific IgG and IgG2b,as compared to controls (Fig.7A and B).We and others [25–27]have previously found that the development of EAMG is not dramatically altered by IFN-cdeficiency.To investigate whether the effects of IL-17on EAMG were related to or depended on IFN-c ,we treated IFN-c –/–mice with IL-17during EAMG induction,using the same regimen used for the B6mice.We found that IFN-c –/–mice and controls responded with elevated anti-AChR antibody production and clinical EAMG at similar magnitudes (Fig.7C and D).These data indicate that IL-17rather than IFN-c influences humoral autoimmune responses in EAMG.IL-17restores EAMG severity of CCL2–/–mice to that of wild-type miceIf the failure to mount autoreactive Th17responses contributes to milder EAMG in the CCL2–/–mice,provision of exogenous IL-17to CCL2–/–mice should reverse this phenotype.To test this hypothesis,we treated CCL2–/–mice with IL-17or PBS attheFigure L2controls homing of IL-6-producing CD11b +cells.B6and CCL2–/–mice were immunized with AChR/CFA to induce EAMG and sacrificed on day 7–14p.i.(A)Production of IL-6by CD4+cells,NK1.1+cells,CD11c +cells and CD11b +cells from B6mice was quantified by intracellular IL-6staining.(B)IL-6released from draining lymph nodes was measured in B6mice as well as CCL-2–/–mice by ELISA (n =4–7each/group).(C,D)Single-cell suspensions of spleen were stained with PE-Cy7-labeled anti-mouse CD11b and allophycocyanin-labeled anti-mouse CD11c before analysis by FACSAria using Diva software.Results represent three independent experiments with reproducible outcomes.**p <0.01.time of induction of pared with PBS-treated CCL2–/–mice that exhibited milder EAMG,AChR-primed CCL2–/–mice treated with IL-17exhibited early onset of muscular weakness (median day of onset:13Æ2vs .27Æ4)that progressed to severe weakness over time (maximum severity 2.6Æ0.3vs .1.5Æ0.2,Fig.7E).Treatment with IL-17elicited higher anti-AChR IgG and prominent IgG2b responses (Fig.7F).These data indicate that defective Th17responses contribute to the reduced severity of muscular weakness in the CCL2–/–mice.Autoreactive Th17cells drive IgG2b antibody response in vivoTransfer of myasthenogenic lymphocytes from humans with MG or from EAMG mice results in clinical EAMG [28,29].We isolated CD4+cells from control mice and CCL2–/–for transfer into RAG1–/–deficient mice (which are devoid of intrinsic T and B cells)[30].The transferred CD4+cells preferentially produced Th17in control mice but not in CCL2–/–mice.Naive B220+B cells were co-transferred into the RAG1–/–recipient mice.At 2weeks following immunization with AChR/CFA,production of anti-AChR IgG and IgG isotypes were evaluated.Mice receiving T cells from CCL2–/–mice had marginally reduced levels of total anti-AChR IgG and significantly reduced levels of IgG2b (Table 1),compared to mice receiving T cells from control EAMG mice.Mice receiving T cells from CCL2–/–mice scarcely developed behavioral signs of EAMG (Table 1).In control experiments,transferred control CD4+cells maintained the Th17phenotype in the recipient mice (percentages of IL-17+CD4+cells:7.2Æ1.6%(control)vs . 1.3Æ0.2%(CCL2–/–),p <0.01).Collectively,these data suggest that auto-reactive Th17cells help B cells to produce pathogenic antibodies,particularly the IgG2b subtype.IL-17-deficient mice develop mild EAMGTo further elucidate the role of IL-17/Th17in EAMG,we immunized control mice and IL-17–/–mice with AChR and CFA and monitored the development of pared to control mice,IL-17–/–mice exhibited significantly milder EAMG,as reflected by evaluation of muscular weakness (Table 2).Produc-tion of IgG2b was impaired in the IL-17–/–mice (Table 2).Table L2–/–CD4+T cells are poor helpers for B cells a)Source of T cells No.of T cells No.of mice/group Disease inci-dence (%)Loss of muscle AChR Mean maximal severity of EAMG (ÆSD)Anti-AChR IgG b)(OD 405)Anti-AChR IgG2b b)(OD 405)B65Â1066(6/6)10053.3Æ12.7% 1.87Æ0.22 1.84Æ0.23 1.21Æ0.20CCL2–/–5Â1067(1/7)14.318.4Æ9.6%0.13Æ0.07*1.03Æ0.300.31Æ0.06*a)CD4+cells were enriched and purified from AChR and CFA primed control mice and CCL2–/–mice and were co-transferred with 5Â106B220+B cells into RAG1–/–recipient mice.b)Muscle weakness was monitored for 55days following cell transfer,sera were collected at the termination of experiments for anti-AChR antibody ELISA.Loss of muscle AChR was quantified by RIA.*p <0.05.Figure 6.AChR-primed IL-6–/–mice fail to mount Th17responses.B6and IL-6–/–were immunized with AChR/CFA as described in Fig.1.Mice were sacrificed on day 7–14p.i.Production of IL-17and IFN-c by cultured spleen cells in response to AChR stimulation (10l g/mL)was measured by intracellular cytokine staining.(A)Expression of CD4vs.intracellular IL-17(upper panel)and IFN-c (lower panel)was compared within this gate.Representative plots are from one of three experiments using 2–3mice/group.(B,C).CD4+cells expressing IFN-c or IL-17in control mice and IL-6–/–mice (n =4–6/group).*p <0.05.DiscussionThis study implicates Th17responses in B cell-dependent (auto)immunity,and establishes a novel role for CCL2in the development of Th17responses.Increasing evidence supports the role of Th17cells in several models of inflammation and autoimmunity.Here we show that the chemokine CCL2,which is elevated during inflammation,is required for the recruitment of IL-6-producing CD11b +cells involved in the generation of autoreactive Th17cells in EAMG.Our model implies that in the lymph node CD11b +APC can produce IL-6to polarize Th cells toward a Th17phenotype,and then the Th17cells can provide help to B cells for the production of pathogenic IgG2b antibodies mediating functional blockade at the neuromuscular junction.It has long been known that autoimmune diseases such as EAMG are associated with elevated production of proinflamma-Table 2.IL-17–/–mice exhibit mild EAMG with reduced incidence a)MiceNo.of mice/group Disease inci-dence (%)Loss of muscle AChR Mean maximal severity of EAMG (ÆSD)Anti-AChR IgG (OD 405)Anti-AChR IgG2b (OD 405)B68(7/8)88%47.6Æ9.2% 1.64Æ0.33 1.91Æ0.34 1.35Æ0.41IL-17–/–4(0/4)010.0Æ2.3%0.09Æ0.05**0.94Æ0.200.22Æ0.03**a)Mice were immunized with AChR and CFA.Muscle weakness was monitored for 35days following the immunization,and sera were collected at this time for anti-AChR antibody ELISA.Loss of muscle AChR was quantified by RIA.**p <0.01.Figure 7.Role of IL-17in EAMG.Groups of B6(A,B),IFN-c –/–(C,D)and CCL-2–/–(E,F)mice were immunized with AChR/CFA and received IL-17or PBS starting at the date of immunization (see Material and methods ).Determination of development of EAMG (A,C,E)and production of anti-AChR antibodies (B,D,F)(n =8/group).*p <0.05,**p <0.01.tory mediators by several cell types.However,the understanding of the intermediate events between immunogenic exposure to autoantigen and subsequent downstream activation of pathogenic immune response has been limited.We found that CCL2-deficient mice developed milder behavioral and histological disease following the induction of EAMG.This result was surprising because monocyte infiltrates,the hallmark of CCL2action,are not prominent in EAMG muscle[24]. This defect was not caused by impaired function of autoreactive B cells,which remained intrinsically intact in the absence of CCL2. However,their capacity to produce anti-AChR IgG antibody,in particular IgG2b,was drastically reduced following immunization. Incidentally,IgG2b is highly myasthenogenic[31].The involvement of CCL2in several experimental models of inflammation and autoimmunity is well established,and appears to partly depend on the ability of this chemokine to direct the inflammatory influx to target organs and to promote Th1and/or Th2cells[10–13].The autoimmune model of EAMG differs from the other systems studied for at least two reasons.First,the muscular weakness characteristic of EAMG is primarily mediated by anti-AChR IgG antibodies produced by autoreactive B cells[19]. Second,because the inflammatory infiltrates are sparse and unrelated to the neuromuscular junctions in EAMG,their contribution to muscular weakness,if any,is likely minute[19]. As such,the B cell-mediated autoimmune process of EAMG, associated with limited inflammation,may represent a system with unique characteristics for studying the immune role of CCL2 independently of inflammation in tissue.We report that the regulation of the immune response to AChR by CCL2is sufficient to alter the magnitude of the symptoms of EAMG,yet CCL2does not directly affect the function of autoreactive B cells in EAMG,as indicated by the fact that upon BCR-cross-linking,CCL2–/–B cells proliferated efficiently,differ-entiated into plasma cells,and released antibodies at a level comparable with controls.Rather,an altered function of Th17cells was most likely responsible for the observed impaired autoanti-body response.CCL2has been reported to affect both Th1and Th2cells[11, 23].We add to this list Th17cells.We think that since we used B6 mice,which are a Th1-prone strain(and immunization with CFA further drove Th1responses),CCL2might have had limited effects on Th2cells in this system.On the other hand,the production of autoantibodies to AChR is clearly a T cell-driven process,which must rely in part on Th1responses,given our finding of preferential IgG2b autoantibodies[32].Although earlier work had indicated that ectopic expression of IFN-c at the neuromus-cular junction provoked MG-like disease[33],our current understanding of the role of IFN-c in EAMG is far from conclusive. For example,mice deficient of IFN-c[25]or IFN-c receptor[26] are resistant to EAMG,yet some groups have shown that IFN-c deficiency may not significantly alter the course of disease[27, 34].Thus,while Th1responses may possibly play a role in the disease depending on the experimental system used,the question arises on whether other inflammatory cytokine(s)with potent pro-pathogenic effects,such as IL-17,are required for the expression of EAMG.We found that AChR-primed CCL2–/–mice failed to mount an effective Th17response,which correlated with an impaired anti-AChR IgG2b response associated with milder disease.The failure to mount productive Th17responsiveness most likely came from impaired homing of the IL-6-producing CD11+cells to the secondary lymphoid organs,as CCL2–/–T cells maintained the capacity to produce IL-17after stimulation.Also,AChR-primed IL-6–/–mice had significantly reduced autoantigen-reactive Th17 responses,and IL-17injection enhanced both the humoral response to AChR and clinical EAMG in B6mice as well as in the IFN-c–/–mice.Finally,CCL2–/–CD4+T cells failed to help B cells to produce disease inducing IgG2b.It has been reported that IL-6–/–mice had impaired anti-AChR IgG2b response upon immunization with AChR[24].The question arises as whether the impaired IgG2b response is due to the lack of IL-6,or altered IL-17response in IL-6–/–mice,or both.Injection of IL-17can partially restore the capacity to mount an IgG2b response in IL-6–/–mice and reestablish susceptibility to the induction of EAMG in these mice.These results emphasized the importance of IL-17in the generation of IgG2b response and the expression of clinical EAMG.Th17cells express CCR2[35].It is plausible that abnormal migration of Th17cells in the CCL2–/–mice also contributes to the mild muscular weakness in this strain.This possibility is currently under investigation.We have not examined the development of EAMG in CCR2–/–mice.Several published works have shown that CCR2–/–mice are resistant to experimental autoimmune diseases,including the B cell-dominant lupus model[36,37].These observations support our current findings and emphasize the importance of CCR2-CCL2 pathway in the development of autoimmunity.The present study focused on the role of CCL2in recruiting IL-6-producing cells capable of promoting generation of auto-reactive Th17responses in B cell-dependent(auto)immunity.We found,further,that Th17plays a central role in humoral immunity in EAMG.We propose that inflammation associated with immunization recruits CD11b+mononuclear phagocytes to lymph nodes,a phenomenon that depends on CCL2.In the lymph nodes of wild-type mice,APC present the autoantigen to Th0cells and make IL-6,in conjunction with other cytokines including TGF-b and IL-23,which polarizes Th cells toward a Th17phenotype.The Th17cells are then required as helpers for the production of high titers of pathogenic anti-AChR IgG2b antibodies from B cells.In the light of new finding by Hsu et al.[38],IL-17may also promote the development of germinal centers.Antibodies to AChR are ultimately responsible for the block at the neuromuscular junction that leads to muscular weakness.It will be important to address next whether CCL2may be a specific therapeutic target to modulate clinical MG.MiceC57BL/6(B6)mice deficient of MCP-1(MCP-1–/–)[11],IL-6 (IL-6–/–),IFN(IFN-c–/–)and RAG(RAG-c–/–)[30]were purchased from The Jackson Laboratory(Bar Harbor,ME).IL-17–/–mice were provided by Dr.Yoichiro Iwakura[5].All mutant mice had been backcrossed to those of the B6background for at least ten generations.Only female mice of7–8weeks of age(at the initiation of the experiments)were used.All mice were bred and maintained in pathogen-free conditions at the animal facilities of the Barrow Neurological Institute in accordance with the Institutional Animal Care and Use Committee regulations.Antigens and peptidesAChR was purified from the electric organs of Torpedo californica (Pacific Biomarine,Venice,CA)by affinity chromatography on a a-cobratoxin-agarose resin(Sigma,St.Louis,MO)[39,40].The isolated product was pure as judged by SDS-PAGE.The AChR a146–162(LGIWTYDGTKVSISPES)peptide was synthesized by Biosynthesis Inc.(Lewisville,TX).Induction,clinical and laboratory evaluation of EAMGMice were injected subcutaneously(s.c.)between the shoulders and back with20l g AChR in CFA in a total volume of100l L. After1month,all mice were boosted once with20l g AChR in IFA s.c.at four sites on shoulders and thighs[38,39].The mice were observed every other day in a blinded fashion for signs of muscle weakness characteristic of EAMG.Clinical manifestations of EAMG were graded from0to3[39]:0,no definite muscle weakness;1+, normal strength at rest but weak with chin on the floor and inability to raise the head after exercise consisting of 20consecutive paw grips;2+,as grade1+and weakness at rest; and3+,moribund,dehydrated,paralyzed.To verify the myasthenic nature of the weakness,we injected i.p.edrophonium chloride(Reversol;Organon,West Orange,NJ),a cholinesterase inhibitor that immediately increases the strength of mice that have clinical EAMG.To further verify the extent of EAMG,we quantified the muscle AChR content by radioimmunoassay using125I-labeled a-bungarotoxin,(Amersham Corp.,Arlington Heights,IL)[27].IL-17injectionRecombinant murine IL-17was purchased from eBioscience(San Diego,CA).At EAMG induction,mice received daily1l g IL-17(in PBS)/mouse i.p.for7consecutive days.Control mice received equal volumes of PBS.Measurement of anti-AChR IgG antibodiesAnti-murine AChR IgG antibodies and anti-torpedo AChR IgG antibodies were detected by ELISA as described previously[39, 40].Briefly,microtiter plates(Corning Glass Works,Corning,NY) were coated with100l L/well murine AChR(0.5l g/mL)or torpedo AChR(2l g/mL)at4 C overnight.After blocking with 10%FBS,serum samples were added and incubated for2h at room temperature.Plates were then incubated for2h with biotinylated rabbit anti-mouse IgG,IgG1,IgG2a,and IgG2b (Invitrogen,Carlsbad,CA),followed by alkaline phosphatase-conjugated ABC reagent(Dakopatts;R&D systems,Minneapolis, MN),and color developed with p-nitrophenyl phosphate.Results were expressed as OD at405nm.CCL2ELISAThe s.c.injection sites and draining lymph nodes were removed from sacrificed control mice and CCL2–/–mice,weighed, homogenized in lysis buffer(PBS with1mM PMSF,0.01mg/ mL aprotinin,and0.01mg/mL leupeptin),sonicated,and centrifuged.The supernatant was assayed for CCL2immunor-eactivity by ELISA(Quantikine;R&D systems,Minneapolis,MN) following the manufacturer's instructions[12].B cell proliferation and differentiationTo determine the extent of B cell proliferation,105purified CD20+CD27+B cells were cultured in96-well round-bottom plates and stimulated with24l g/mL goat-anti-mouse IgM alone or in combination with5l g/mL anti-mouse CD40[41].After 3–5days of culture,[3H]thymidine was added to the culture for additional18h.[3H]Thymidine uptake was measured using a liquid scintillation counter.In some experiments,stimulated and control cultures were stained after various intervals with anti-CD19PerCP-Cy5.5(1D3)and allophycocyanin-conjugated anti-CD138(281–2)to visualize plasma cells,which were identified as CD19low/–CD138+cells[42].Plasma cell differentiation in AChR-primed control mice and CCL2–/–micePurified B cells from lymph nodes were either left unstimulated or stimulated with a combination of anti-CD40and IgM.After6days of culture,B cells were identified as CD19+and plasma cells as CD19low/–CD138+.。

13455143_复方忍冬藤提取物促进骨折愈合及抗炎作用研究

中国畜牧兽医　２０１７，４４（１）：２７５２８１犆犺犻狀犪犃狀犻犿犪犾犎狌狊犫犪狀犱狉狔牔犞犲狋犲狉犻狀犪狉狔犕犲犱犻犮犻狀犲ｄｏｉ：１０．１６４３１／ｊ．ｃｎｋｉ．１６７１７２３６．２０１７．０１．０３９复方忍冬藤提取物促进骨折愈合及抗炎作用研究贾海燕，滕旭东，王　慧，张慧珍（临沂大学药学院，临沂２７６０００）摘　要：试验旨在探讨复方忍冬藤提取物对骨折愈合和抗炎的作用。

采用牙科电钻制备兔右侧桡骨骨折模型，通过测定血清中钙、磷和碱性磷酸酶的含量，探明其对骨折家兔血清生化指标的影响。

利用ＬＰＳ诱导的ＲＡＷ２６４．７细胞建立炎症模型，采用ＥＬＩＳＡ法测定复方忍冬藤提取物对ＬＰＳ诱导的ＲＡＷ２６４．７细胞释放的炎症因子ＩＬ６、ＴＮＦ α、ＩＬ１β和ＮＯ含量的变化。

结果显示，利用牙科电钻可成功制备兔骨折模型，复方忍冬藤提取物可增加骨折家兔血清中钙和碱性磷酸酶的水平，降低血清中磷含量。

与ＬＰＳ组相比，５０～２００μｇ／ｍＬ复方忍冬藤提取物对ＬＰＳ诱导的ＲＡＷ２６４．７细胞分泌的炎症因子ＴＮＦ α、ＩＬ１β和ＮＯ的含量具有显著的抑制作用（犘＜０．０５），对ＩＬ６的含量无显著影响（犘＞０．０５）。

综上所述，复方忍冬藤提取物对骨折愈合有促进作用，并具有较好的抗炎效果。

关键词：复方忍冬藤；提取物；骨折愈合；抗炎中图分类号：Ｓ８５８．２９１文献标识码：Ａ文章编号：１６７１７２３６（２０１７）０１０２７５０７收稿日期：２０１６０５２３基金项目：国家级大学生创新创业训练计划项目（２０１５１０４５２０２５）；山东省科技发展计划项目（２０１３ＧＳＦ１１９０９）作者简介：贾海燕（１９９４），女，菏泽鄄城人，学士，研究方向：中草药提取及药效，Ｅｍａｉｌ：８１９３６７４０４＠ｑｑ．ｃｏｍ通信作者：王　慧（１９８４），女，山东泗水人，博士，副教授，研究方向：中药药理与毒理学，Ｅｍａｉｌ：ｗａｎｇｈｕｉ０５１２＠ｌｙｕ．ｅｄｕ．ｃｎ犈犳犳犲犮狋狊狅犳犕犻狓犲犱犆犪狌犾犻狊犔狅狀犻犮犲狉犪犲犼犪狆狅狀犻犮犪犲犈狓狋狉犪犮狋狊狅狀犉狉犪犮狋狌狉犲犎犲犪犾犻狀犵犪狀犱犃狀狋犻犻狀犳犾犪犿犿犪狋犻狅狀ＪＩＡＨａｉｙａｎ，ＴＥＮＧＸｕｄｏｎｇ，ＷＡＮＧＨｕｉ，ＺＨＡＮＧＨｕｉｚｈｅｎ（犛犮犺狅狅犾狅犳犘犺犪狉犿犪犮狔，犔犻狀狔犻犝狀犻狏犲狉狊犻狋狔，犔犻狀狔犻２７６０００，犆犺犻狀犪）犃犫狊狋狉犪犮狋：Ｔｈｉｓｅｘｐｅｒｉｍｅｎｔｗａｓａｉｍｅｄｔｏｓｔｕｄｙｔｈｅｅｆｆｅｃｔｓｏｆｍｉｘｅｄ犆犪狌犾犻狊犔狅狀犻犮犲狉犪犲犼犪狆狅狀犻犮犪犲ｅｘｔｒａｃｔｓｏｎｆｒａｃｔｕｒｅｈｅａｌｉｎｇａｎｄｉｎｆｌａｍｍａｔｉｏｎ．Ｒａｂｂｉｔｒｉｇｈｔｒａｄｉｕｓｆｒａｃｔｕｒｅｍｏｄｅｌｗａｓｉｎｄｕｃｅｄｂｙｄｅｎｔａｌｄｒｉｌｌｉｎｏｒｄｅｒｔｏｓｔｕｄｙｔｈｅｍｅｄｉｃｉｎｅ’ｓｅｆｆｅｃｔｏｎｆｒａｃｔｕｒｅｈｅａｌｉｎｇ．Ｍｏｒｅｏｖｅｒ，ｔｈｅｃｏｎｔｅｎｔｓｏｆＣａ，Ｐａｎｄａｌｋａｌｉｎｅｐｈｏｓｐｈａｔａｓｅｉｎｓｅｒｕｍｗｅｒｅｍｅａｓｕｒｅｄｔｏｅｘｐｌｏｒｅｔｈｅｉｎｆｌｕｅｎｃｅｓｏｆｍｉｘｅｄ犆犪狌犾犻狊犔狅狀犻犮犲狉犪犲犼犪狆狅狀犻犮犪犲ｅｘｔｒａｃｔｓｏｎｓｅｒｕｍｂｉｏｃｈｅｍｉｃａｌｉｎｄｅｘｅｓｏｆｆｒａｃｔｕｒｅｄｒａｂｂｉｔｓ．ＲＡＷ２６４．７ｃｅｌｌｗａｓｉｎｄｕｃｅｄｂｙＬＰＳｉｎｏｒｄｅｒｔｏｒｅｓｅａｒｃｈｔｈｅｅｆｆｅｃｔｏｆｍｅｄｉｃｉｎｅｏｎｉｎｆｌａｍｍａｔｉｏｎ．ＴｈｅｃｏｎｃｅｎｔｒａｔｉｏｎｓｏｆＴＮＦ α，ＩＬ１β，ＩＬ６ａｎｄＮＯｗｅｒｅｍｅａｓｕｒｅｄｂｙＥＬＩＳＡ．Ｔｈｅｒｅｓｕｌｔｓｓｈｏｗｅｄｔｈａｔｔｈｅｒａｂｂｉｔｆｒａｃｔｕｒｅｍｏｄｅｌｗａｓｓｕｃｃｅｓｓｆｕｌｌｙｐｒｅｐａｒｅｄｂｙｄｅｎｔａｌｄｒｉｌｌ，ａｎｄｔｈｅｅｘｔｒａｃｔｓｏｆ犆犪狌犾犻狊犔狅狀犻犮犲狉犪犲犼犪狆狅狀犻犮犪犲ｃｏｕｌｄｉｎｃｒｅａｓｅｔｈｅｌｅｖｅｌｓｏｆＣａａｎｄａｌｋａｌｉｎｅｐｈｏｓｐｈａｔａｓｅａｎｄｒｅｄｕｃｅｔｈｅＰｃｏｎｔｅｎｔｉｎｔｈｅｓｅｒｕｍｏｆｆｒａｃｔｕｒｅｄｒａｂｂｉｔｓ．ＴｈｅＴＮＦ α，ＩＬ１βａｎｄＮＯｌｅｖｅｌｓｉｎＲＡＷ２６４．７ｃｅｌｌｉｎｄｕｃｅｄｂｙＬＰＳｗｅｒｅｓｉｇｎｉｆｉｃａｎｔｌｙｉｎｈｉｂｉｔｅｄｂｙｔｈｅｅｘｔｒａｃｔｓｗｉｔｈｃｏｎｃｅｎｔｒａｔｉｏｎｓｒａｎｇｉｎｇｆｒｏｍ５０ｔｏ２００μｇ／ｍＬ（犘＜０．０５）．Ｈｏｗｅｖｅｒ，ｔｈｅｅｘｔｒａｃｔｓｈａｄｎｏｓｉｇｎｉｆｉｃａｎｔｅｆｆｅｃｔｏｎｔｈｅｌｅｖｅｌｏｆＩＬ６（犘＞０．０５）．Ｔｈｅｓｅｒｅｓｕｌｔｓｄｅｍｏｎｓｔｒａｔｅｄｔｈａｔｔｈｅｅｘｔｒａｃｔｓｏｆｍｉｘｅｄ犆犪狌犾犻狊犔狅狀犻犮犲狉犪犲犼犪狆狅狀犻犮犪犲ｃｏｕｌｄｐｒｏｍｏｔｅｔｈｅｆｒａｃｔｕｒｅｈｅａｌｉｎｇａｎｄｐｏｓｓｅｓｓｅｄｇｏｏｄｒｅｓｉｓｔａｎｃｅｉｎｆｌａｍｍａｔｉｏｎｅｆｆｅｃｔ．犓犲狔狑狅狉犱狊：ｍｉｘｅｄ犆犪狌犾犻狊犔狅狀犻犮犲狉犪犲犼犪狆狅狀犻犮犪犲；ｅｘｔｒａｃｔｓ；ｆｒａｃｔｕｒｅｈｅａｌｉｎｇ；ａｎｔｉｉｎｆｌａｍｍａｔｉｏｎ忍冬藤（犆犪狌犾犻狊犔狅狀犻犮犲狉犪犲犼犪狆狅狀犻犮犪犲）为忍冬科（Ｃａｐｒｉｆｏｌｉａｃｅａｅ）植物忍冬（犔狅狀犻犮犲狉犪犼犪狆狅狀犻犮犪Ｔｈｕｎｂ．）的干燥藤茎，具有清热解毒、抗炎止痛、活血化瘀、通经活络的功效［１２］，可作为一种安全、可开发的抗炎药物［３］。

化学专业英语3.12翻译

selective precipitation选择性沉淀
IF语句的应用
3.12Additional Aspects of Aqueous Equilibria
The phenomenon of lowering the degree of ionization
of a weak electrolyte by adding a solution of a strong
剂是一种与其共轭碱或酸有一种不同颜色的弱酸或碱。在滴定曲线中一种强酸强碱的滴定的pH在等当点附近呈现出一个巨大的变化。强酸弱碱或弱酸强碱滴定，在等当点附近的pH值的变化不是很大bility equilibrium exists when a chemical compound in the solid state is in chemical equilibrium with a solution of that compound. The equilibrium is an example of dynamic and heterogeneous equilibrium in that some individual molecules migrate between the solid and solution phases such that the rates of dissolution and precipitation are equal to one another. The Solubility-product constant(or simply the solubility product),Ksp, is an equilibrium constant that expresses quantitatively the extent to which the compound dissolves. The Ksp is equal to the product of the concentrations of the ions involved in the equilibrium, each raised to the power of its coefficient in the equilibrium equition. The value of Ksp can be obtained from the solubility, and vice versa. Substances that have Ksp<<1 are classified as insoluble. Salts that have Ksp, between 10-2 and 10-5 are slightly soluble , and solids which have Ksp>10-2 are soluble. The solubility of a slightly soluble substance can be decreased by the presence of a common ion. The solubility of compounds containing basic anions increase as pH decreases. Salts with anions of negligible basicity (the anions of strong acids ) are unaffected by pH changes.

Clinical aspects and biological bases of drug-resistant epilepsies

Epilepsy Research 34(1999)109–122Clinical aspects and biological bases of drug-resistant epilepsiesGiovanni Regesta a,*,Paolo Tanganelli baDepartment of Neurology ,Epilepsy Center ,San Martino Hospital ,Viale Benedetto XV ,10-16132Geno 6a ,ItalybDepartment of Neurology ,P .A .Micone Hospital ,Geno 6a ,Italy Received 11February 1998;received in revised form 22September 1998;accepted 30September 1998AbstractThe deﬁnition of drug-resistant epilepsy (DRE)is elusive and still controversial owing to some unresolved questions such as:how many drugs should be tried before a patient is considered intractable;to which extent side-effects may be acceptable;how many years are necessary before establishing drug resistance.In some cases,the view of epilepsy as a progressive disorder constitutes another important issue.Despite the use of new antiepileptic drugs (AEDs),intractable epilepsy represents about 20–30%of all cases,probably due to the multiple pathogenetic mechanisms underlying refractoriness.Several risk factors for pharmacoresistance are well known,even if the list of clinical features and biological factors currently accepted to be associated with difﬁcult-to-treat epilepsy is presumably incomplete and,perhaps,disputable.For some of these factors,the biological basis may be common,mainly represented by mesial temporal sclerosis or by the presence of focal lesions.In other cases,microdysgenesis or dysplastic cortex,with abnormalities in the morphology and distribution of local-circuit (inhibitory)neurons,may be responsible for the severity of seizures.The possible inﬂuence of genes in conditioning inadequate intraparenchimal drug concentration,and the role of some cytokines determining an increase in intracellular calcium levels or an excessive growth of distrophic neurites,constitute other possible mechanisms of resistance.Several hypotheses on the mechanisms involved in the generation of DRE have been indicated:(a)ontogenic abnormalities in brain maturation;(b)epilepsy-induced alterations in network,neuronal,and glial properties in seizure-prone regions such as the hippocampus;(c)kindling phenomenon;(d)reorganization of cortical tissue in response to seizure-induced distur-bances in oxygen supply.Such hypotheses need to be conﬁrmed with suitable experimental models of intractable epilepsy that are speciﬁcally dedicated,which have until now been lacking.©1999Elsevier Science B.V.All rights reserved.Keywords :Epilepsy;Drug resistance;Biological bases;Clinical aspects;Genetic factors1.IntroductionTo investigate the clinical aspects and the bio-logical bases of drug-resistant epilepsies (DRE)is*Corresponding author.Tel.:+390105552542;fax:+390105556603.e-mail :divneurologia@smartino.ge.it.0920-1211/99/$-see front matter ©1999Elsevier Science B.V.All rights reserved.PII:S 0920-1211(98)00106-5G.Regesta,G.Regesta/Epilepsy Research34(1999)109–122 110a complex problem and it presupposes,ﬁrst of all,a deﬁnition of DRE.However,a universally accepted deﬁnition of DRE does not yet exist due to some unsolved controversial issues which impede attaining a consensus about this matter. According to Theodore(1993),we have to ad-mit that current deﬁnitions of uncontrolled seizures areﬂuid.Hauser(1992)remarks that ‘‘The deﬁnition of drug resistant epilepsy is elu-sive and may vary with the question being asked,upon the investigator’s interest and avail-able procedures.In some ways,all epilepsy is drug resistant in that there is no evidence that action of anti epileptic drugs(AEDs)is other than palliative(preventing seizures),but without effect on the underlying pathologic state.’’Cas-cino(1990)considers the seizures persisting in-tractable,despite maximally tolerated mono-therapy or combination AED therapy.Similarly, Juul-Jensen(1986)deﬁnes the presence of re-peated seizures during years in a patient receiv-ing appropriate treatment at high doses as refractory epilepsy.All these deﬁnitions,although useful for clini-cal purposes,are not based on objective,clearly quantiﬁed criteria of assessment,and therefore do not permit comparison of the different stud-ies.Furthermore,they focus on the persistence of seizures as the sole index of refractoriness, not taking into account the patient’s ability to function in everyday life and psychosocial prob-lems related to epilepsy or therapy.This view is pointed out in the deﬁnition of Schachter (1993):a patient with refractory epilepsy is one who is unable to lead a lifestyle consistent with his capabilities because of seizures,AED side-ef-fects,and/or psychosocial problems.The persisting uncertainty about the deﬁnition and,consequently,the impossibility of exactly identifying these subjects,produces some impor-tant consequences,mainly concerning the selec-tion of cases to be considered for surgery and the unavoidable heterogeneity of patients in-cluded in clinical trials of new AEDs.In theﬁrst part of this paper,we will try to identify and discuss the issues hampering the at-tainment of a clear-cut deﬁnition of DRE.In the second part,the clinical features and the biological factors more frequently encountered in difﬁcult-to-treat subjects will be analyzed.2.Issues in deﬁning DREThere is no doubt that,when dealing with the problem of pharmacoresistance,one should be aware of the possibility of a false pharmacoresis-tance,a not always easily recognizable phe-nomenon that may be found in all chronic diseases.An exhaustive analysis of this important matter is beyond the scope of this article;how-ever,it may be useful to summarize the main causes of pseudo-refractory epilepsy.1.Diagnostic errors leading to inappropriatedrug selection:e.g.ethosuximide in complex partial seizures(CPS)misdiagnosed as ab-sences,carbamazepine or phenytoin,in juve-nile myclonic epilepsy(JME)misdiagnosed as grand mal(GM)epilepsy,etc.2.Inappropriate assessment of response:pseudo-seizures developing as a substitute for epileptic seizures,paradoxical drug intoxication.3.Suboptimal dosing regimen:drug not tried atthe highest tolerated dosage,inappropriate dosing frequency.4.Inappropriate patient behaviour:poor compli-ance,detrimental lifestyle.Coming back to the difﬁculty in deﬁning and assessing DRE,some controversial issues and un-solved questions may be identiﬁed.2.1.How many years are needed before establishing drug resistance?The duration of active epilepsy before a patient might be labelled as pharmacoresistant is much debated.It is widely accepted that about60–70% of people with epilepsy will achieve remission of seizures in a variable,generally short,period of time.The duration of active epilepsy before a patient might be labelled as pharmacoresistant is much debated.For example,Leppik(1992)pro-poses that DRE can be deﬁned as seizures not completely controlled with AEDs1year after onset,while Loyning and Hauglie-Hanssen(1995)G.Regesta,G.Regesta/Epilepsy Research34(1999)109–122111suggest at least2years of adequate drug trials with at least two drugs for establishing refrac-toriness.However,it should be considered that, in some cases,even after10years,seizure con-trol may be obtained(Hauser,1992). Furthermore,the rate of remission and the time needed for achieving it are rather different when data derived from hospital-and commu-nity-based studies are considered.Consequently, in order to answer this question,we have to consider the prognosis of the individual cases,in relation to the speciﬁc type of seizures and/or of epileptic syndrome.As regards outcome,the epileptic syndromes have been classiﬁed into four different prognostic groups(Sander,1993). Following this grouping,the time required for establishing drug resistance cannot be an abso-lute value but it is related to the belonging group of the syndrome,as,for example,a case of childhood absence epilepsy not responding to treatment in the short term.Unfortunately,this classiﬁcation does not of-fer an answer in any case.In fact,while some epileptic syndromes are clear cut,for most of them a disagreement about their precise limits exists,with possible overlaps.The possibility of different etiologies for the same syndrome,and the existence of cases not falling into the recog-nized syndromes,must also be considered,to-gether with the statement that sometimes a syndromic diagnosis is made retrospectively.For these reasons,a better-deﬁned answer requires further prospective studies focusing on the prog-nosis of speciﬁc epileptic syndromes(Sander, 1993).2.2.When should a patient be considered to ha6e failed on the initially prescribed drug,and how many drugs should be tried before his/her epilepsy might be regarded as intractable?The score suggested by Schmidt(1986)for the assessment of treatment failures,admits only the possibility of primary drugs in monotherapy and does not take into account the number of drugs to be tried(Table1).On the other hand,a cer-tain percentage( 40%)of patients not ade-quately controlled with a single drug treatment Table1Medical intractability scale(Schmidt,1986)Index of in-Seizures persist despite the followingtreatmenttractability0Other than primary drug regardless ofits daily dose1Primary drug below the recommendeddaily dose2Primary drug within the recommendeddaily dose3Primary drug with plasma concentra-tions within the recommended‘thera-peutic range’4Primary drug with maximum tolerabledaily dose5More than one primary drug withmaximum tolerable daily dose in sub-sequent single-drug therapy for atleast9monthsmay enter remission with a combination of two or three AEDs(Mattson,1994).A grading system for evaluating the probabil-ity of achieving future seizure freedom,based on the failure of the appropriate treatment with one or more drugs,has been proposed by Perucca (1996)(Table2).2.3.Should onlyﬁrst-line treatments or also second-line treatments be tried before establishing drug resistance?When initiating a treatment,the selection of an AED is currently based on its expected efﬁcacyTable2Grading system for drug resistance(Perucca,1996)DeﬁnitionGrade Probability of futureseizure freedom(%)Failure on appropriate530–351treatment with one drug510–152Failure with two drugsgiven alone or com-bined355Failure with three drugsgiven alone or com-binedG.Regesta,G.Regesta/Epilepsy Research34(1999)109–122 112for speciﬁc seizure type(s)and epilepsy.There-fore,therapeutic algorithms suggestingﬁrst-line and second-line drugs according to the diagnosis have been proposed.However,except for rare situations,these recommendations are not based on irrefutable data and it is well known that different drugs may show a corresponding efﬁ-cacy in a given seizure type or -parative monotherapy trials in partial and secondarily generalized tonic–clonic seizures found no conclusive difference in efﬁcacy among carbamazepine,phenytoin,phenobarbital,and valproate(Mattson et al.,1985,1992). Comparing the efﬁcacy of the new AEDs is even more uncertain because studies concerning their use in monotherapy have been limited until now.The results of a metanalysis of efﬁcacy data from randomized placebo-controlled add-on tri-als of all new drugs do not allow an evidence-based choice between these agents(Marson et al.,1997).Therefore,drug selection should be based not only on the speciﬁc antiepileptic action(provided that it is thoroughly known),but also on other factors such as pharmacokinetic properties,ad-verse effects,and patient characteristics.Strictly related to this issue is the vexata quaestio concerning monotherapy vs polyther-apy.In the1980s,after the negative appraisal of polytherapy by Reynolds and Shorvon(1981), polytherapy became a synonym of‘bad therapy’. Monotherapy was therefore considered a ‘dogma’.More recently,this dogma crumbled, due to the evidence of synergistic therapeutic ef-fects of some comedications(Rowan et al.,1983; Bourgeois and Dodson,1989).Other different favourable combinations are still under evalua-tion(Robinson et al.,1993;Stolarek et al., 1993).While the advantages of monotherapy in the initial management of epilepsy are universally ac-cepted,no general agreement exists about the strategy to be used when seizures continue,de-spite administration of a single drug at adequate dosages.Two therapeutic alternatives can be pro-posed for these patients:(a)to add a second drug or(b)to substitute the initial drug with another,known to be active against the same seizure type(Beghi and Perucca,1995;Schmidt and Gram,1995).The main advantages of the latter policy include the possibility of evaluating separately the effects of individual drugs,of re-ducing the risk of toxicity and of avoiding ad-verse drug interactions.On the other hand,a possible disadvantage could be represented by the longer delay in obtaining control of the seizures in those patients responding only to bitherapy,with a potentially unfavourable im-pact on overall prognosis(Reynolds et al.,1983). Except for two studies(Hakkarainen,1980;Tan-ganelli and Regesta,1996),no other trial has yet evaluated the risk/beneﬁt ratio of add-on therapy in patients unresponsive to alternative monother-apy.A rational choice of therapeutic strategies in patients who failed to respond to initial monotherapy is so hampered by lack of relevant information from controlled trials.Thus,a scien-tiﬁc evaluation of different treatment options in epileptic patients who continue to have seizures despite adequate initial monotherapy is desirable.2.4.What is the potential role of AEDs?Recent clinical trials proved new AEDs to be effective in patients refractory to conventional drugs.However,the percentage of seizure-free patients remains to be established,as most stud-ies report only the percentage of patients with signiﬁcant reduction(more than50%)in seizure frequency(Marson et al.,1997).Only a small minority(less than2%)of patients refractory to old drugs is reported to become seizure free with new AEDs(Walker and Sander,1996).The per-sisting lack of an acceptable model of drug-resis-tant seizures represents the most probable explanation for these non-striking results (Lo¨scher,1997).2.5.To which extent should side-effects be considered acceptable?Systemic toxicity and neurotoxicity strongly contribute to medical failure,but it must be con-sidered that the extent to which patients complain about side-effects varies according to the severity of the side-effects and the level of functioning ofG.Regesta,G.Regesta/Epilepsy Research34(1999)109–122113the patient.A widely accepted,standardized as-sessment is lacking.2.6.Should cases with different frequency and/or se6erity of episodes during treatment be considered unitedly in the same way?Usually,all these cases are deﬁned as refrac-tory.The term results here to be quite generic and not appropriate to cover this wide spectrum of situations,whose biological bases are likely to be different.An attempt toﬁx the boundaries has been made by Camﬁeld and Camﬁeld(1996),who consider intractable only the patients presenting at least one seizure every2months in theﬁrst5 years,and for the longer term at least one seizure per year.2.7.Should epilepsy whose seizures relapse after a prolonged period of remission be considered refractory?Even after a long-term remission,a small mi-nority of patients may relapse(10%of patients in the Tunbridge study by Goodridge and Shorvon (1983))and for a part of them the risk exists of becoming intractable(Shorvon,1984).Isolated or sporadic seizures may be frequently related to facilitating situations such as psyco-physical stress,sleep disturbances,etc.,even if their exact role cannot be determined.Obviously, in these cases,the term drug resistance is not justiﬁed.It should be applied,on the contrary,to cases in which the repetitiveness of seizures sug-gests a progression of the epileptogenic process (see later).2.8.The concept of temporary(age-dependent) drug resistancePopulation studies(Camﬁeld and Camﬁeld, 1996)indicate that during long-term follow-up, many children with intractable epilepsy eventually have remission of their seizure disorder.In a retrospective analysis,Huttenlocher and Hapke(1990)found that in145children with medically resistant seizures for2or more years, and with a follow-up ranging from5to20years after seizure onset,remission occurred at rates of 4%per year for children with borderline or nor-mal intelligence and at1.5%per year for retarded children.Except for myoclonic seizures,all other types were equally likely to remit over time.In a prospective study of children with medically in-tractable complex partial seizures of temporal lobe origin,Lindsay et al.(1979)observed that one-third of them became seizure-free adults. This phenomenon of the age-dependent drug resistance is partly related to the natural history of some types of epilepsy and partly to variations with age of some factors,i.e.drug kinetics(Cloyd et al.,1997),glial proliferation,glia/neurons ratio, CSFﬂow,maturation of enzymatic and neuro-transmitter systems(White et al.,1983).2.9.The6iew of some forms of epilepsy as dynamic and/or progressi6e disorders,rather than in6ariably static conditionsThere is little doubt that in many individual patients,epilepsy appears to be either severe or mild at different times,under the inﬂuence of constitutional and environmental factors (Reynolds et al.,1983).On the other hand,it has been suggested that some forms of epilepsy are progressive owing to the assumption that epilep-togenesis is a continuous dynamic process that could make seizures more severe(Engel,1990, 1993;Heinemann et al.,1994).The bases for the susceptibility to progression may be attributed to some factors such as:the epileptogenic potential of the initial insult,topographic variations in epileptogenicity of some areas of the brain,ad-verse effects on cerebral development by early onset seizures,slow pathological changes in chronic epilepsy(re-arrangement of neuronal cir-cuitries in the hippocampus,proliferation of mossyﬁbres in hippocampal sclerosis),and ge-netic predisposition(Jennet1975;Wilmore et al., 1978;Andermann,1982;Okada et al.,1984;En-gel,1990;Kim et al.,1990;Babb,1991;Engel and Shewmon1991;Engel,1992;Wieser et al., 1993).Secondary foci and epilepsy-induced alter-ations in neural network,neuronal,and glial properties may be involved in this progression, mainly as regards temporal lobe and frontal cor-G.Regesta,G.Regesta/Epilepsy Research34(1999)109–122 114tex seizures.Another hypothesis concerns the kindling phenomenon,even if its occurrence in human epilepsy remains to be proven(Heine-mann et al.,1994;Shinnar and Berg,1996).The following query is strictly related to the previous issue.2.10.Would more effecti6e therapy at the onset reduce the risk of intractable epilepsy?It is current practice not to treat after a single seizure.However,in most cases(up to80%), patients will present further attacks,and consid-ering that a negative association between seizure outcome and pre-treatment number of seizures has been consistently reported(Juul-Jensen, 1964a;Rodin,1968;Okuma and Kumashiro, 1981;Elwes et al.,1984;Shorvon and Reynolds, 1986;Van Donselaar et al.,1991;Collaborative Group for the Study of Epilepsy.,1992),this attitude seems questionable.On the contrary, other authors(Shinnar and Berg,1996)attach greater importance to the type rather than the number of seizures.3.Possible predictors of refractorinessApart from these semantic and methodologi-cal considerations,intractable epilepsy consti-tutes a reality regarding some20–30%of all epileptic patients(Annegers et al.,1979; Reynolds et al.,1983;Shorvon,1984)and5–10%of all incidental cases(Hauser,1992).The question arises if predictors of DRE ex-ist.Numerous studies have been performed to identify predictive factors of intractability,with different results depending on the investigator’s interest and the type of population examined. Most studies focus on electroclinical features, some others investigate the morphological as-pects.More recently,molecular and neurochemi-calﬁndings have been reported that could represent a key for understanding the causes of some forms of refractoriness.The identiﬁcation of animal models mimicking patterns of pharmacological resistance of the hu-man epilepsy might represent a useful mean for studying the mechanisms of intractability and for developing more effective drugs(Lo¨scher, 1997).3.1.Electroclinical predictorsSeveral factors have been associated with in-tractability,mostly in relation to focal epilepsy since about60%of resistant patients suffer from partial seizures.When multiple factors are present in single case,the risk of intractability obviously increases.The most commonly re-ported are as follows.3.1.1.Early seizure onsetThere is general agreement that an early onset of seizures within theﬁrst year of life carries an adverse prognosis,being associated with a high rate of seizure persistence(Annegers et al.,1979; Soﬁjanov,1982).The risk increases if early on-set is combined with high seizure frequency (Duchowny,1987).3.1.2.Length of the history before treatmentFor some authors,length of time fromﬁrst seizure and number of seizures before onset of therapy are unfavourable prognostic factors (Juul-Jensen,1964a;Rodin,1968;Shorvon and Reynolds,1982;Elwes et al.,1984;Van Donse-laar et al.,1991;Collaborative Group for the Study of Epilepsy.,1992).This association is de-nied in other studies(Sillanpa¨a¨,1993;Camﬁeld and Camﬁeld,1996).Shinnar and Berg(1996), reviewing the data from the paper of Shorvon and Reynolds(1986),observe that the outcome was only correlated with the number of complex partial seizures and not with the number of tonic–clonic seizures.Similarly,in a randomized trial comparing two AEDs in drug-naive pa-tients in Kenya,subjects with longer duration of epilepsy and those with a history of more than 100generalized tonic–clonic seizures responded equally well to medication,as did those with epilepsy of briefer duration and fewer seizures (Feksi et al.,1991).3.1.3.High seizure frequencyDaily or weekly seizures constitute a risk fac-G.Regesta,G.Regesta/Epilepsy Research34(1999)109–122115tor for intractability especially in infancy(Ju-ulJensen,1964b;Oller-Daurella et al.,1976; Aicardi,1988;Sillanpa¨a¨,1993;Camﬁeld and Camﬁeld,1996).3.1.4.History of febrile seizuresFebrile seizures,particularly complex febrile seizures and febrile status epilepticus,have been hypothesized as one cause of hippocampal scle-rosis(Falconer et al.,1964;Sagar and Oxbury, 1987;Cendes et al.,1994;French et al.,1994). Yet,the incidence of hippocampal sclerosis fol-lowing childhood febrile seizures is low(Erem-berg and Morris,1987;Duchowny,1997; Offringa et al.,1994)and epidemiologic studies have shown that the prognosis of febrile seizures is generally benign(Nelson and Ellenberg,1978; Consensus Development Panel.,1980;Annegers et al.,1987;Freeman,1990;Verity and Golding, 1991;Camﬁeld and Camﬁeld,1995).Random-ized trials of treatment to prevent febrile seizures provided further evidence against a rela-tionship between febrile seizures and later epilepsy(Wolf and Forsythe,1989;Rosman et al.,1993;Knudsen et al.,1995).3.1.5.Type of seizuresAbout60%of patients with intractable epilepsy suffer from partial seizures(Reynolds et al.,1983),mainly of complex type.The presence of multiple seizure type generally constitutes a predictor of reduced probability of remission (Hauser,1992).However,for other authors,seizure type is not an inﬂuential predictor of intractability (Huttenlocher and Hapke,1990;Sillanpa¨a¨,1993; Camﬁeld et al.,1993),particularly if epilepsy be-gins in infancy.3.1.6.Persistence of seizuresThe longer the seizures continue on treatment, the less likely is remission to occur(Reynolds et al.,1983;Sillanpa¨a¨,1993).In cases of seizures remaining uncontrolled for more than4years, the probability of remission is low(Annegers et al.,1979;Loiseau,1986;Hauser,1992).3.1.7.Status epilepticusOccurrence of status epilepticus constitutes one of the main predictors of intractability for some authors(Sillanpa¨a¨,1993),while for others the prognosis depends also on the underlying cause and the pre-existing neurological status of the patient(Maytal et al.,1989;Shinnar et al., 1992;Hauser,1990;Gross-Tsur and Shinnar, 1993;Dodson et al.,1993;Verity et al.,1993; Towne et al.,1994).Epilepsia partialis continua, mainly type2,generally represents a regional lesion involving the cortex,with poor prognosis in most cases(Rasmussen’s encephalitis).3.1.8.Epilepsy syndromesThe type of epileptic syndrome is important in determining prognosis.However,as previ-ously reported,in a large proportion of patients, a speciﬁc syndrome cannot be identiﬁed at the time of diagnosis and,in many cases,a syn-dromic diagnosis can be made only retrospec-tively(Sander,1993;Shinnar and Berg,1996). In some patients,particularly in those with few seizures,the assignment to a speciﬁc syndrome cannot be made even after several years of treatment(Shinnar and Berg,1996).3.1.9.Abnormal neurological statusPatients with brain damage are less likely to go into remission than do neurologically normal patients(Aicardi,1990;Berg et al.,1995).The more severe the neurologic impairment,the greater the chances of medical intractability(Tr-evathan et al.,1988;Uvenbrandt,1988),particu-larly when associated with high seizure frequency(Chevrie and Aicardi,1979).3.1.10.Electroencephalography abnormalitiesThe signiﬁcance of electroencephalography (EEG)as a predictor of prognosis has been vari-ably interpreted(Rodin,1968;Holowach-Thurston et al.,1982;Todt,1984;Shinnar et al., 1985).Gross EEG abnormality in a patient with epilepsy generally implies a poor prognosis for becoming seizure free(Rodin,1968;Rowan et al., 1980).The presence of multifocal paroxysms would be the only EEG-signiﬁcant risk factor for negative outcome(Satishandra et al.,1987).InG.Regesta,G.Regesta/Epilepsy Research34(1999)109–122 116Table3Structural lesions associated with epilepsy(Vinters et al.,1993) Malformati6eCortical dysplasiaMicrodysgenesis(Meencke)Focal dysplasiaCortical dysplasia with hamartomatous proliferation of neuroectodermal cellsPolymicrogyriaLissencephaly/PachygyriaHemimegalencephalyVascular malformationsArteriovenous malformationCavernous hemangiomaNeoplasticGliomasGangliogliomasMetastatic tumorsDysembryoplastic neuroepithelial tumorOthersFamilial and metabolicWith focal lesions;phacomatosisTuberous sclerosisNeuroﬁbromatosisEncephalotrigeminal angiomatosis,Sturge–Weber disease With diffuse lesionsLysosomal enzyme deﬁcienciesPeroxisomal disordersMitochondrial enzyme disordersUnknown etiology,e.g.Alexander’s disease, lipofuscinosis,myelinopathiesLafora body diseaseMiscellaneous myoclonic epilepsiesCerebro6ascular disease and traumaIschemicHemorrhagicPost-traumaticInﬂammatory/infectiousFulminant encephalitis,e.g.due to herpes virusChronic:e.g.parasiticRasmussen’s encephalitisAmmon’s horn(hippocampal)sclerosis 3.1.11.Family history for epilepsyNo complete agreement exists about the impor-tance of a family history of epilepsy in determining prognosis.For some authors,it may constitute an adverse factor of risk(Satishandra et al.,1987; Shafer et al.,1988;Elwes and Reynolds,1990).3.2.Biological predictorsBiologic factors inﬂuencing prognosis refer al-most exclusively to symptomatic epilepsy that may be the result of a structural lesion including a wide variety of pathologic conditions(inﬂamma-tory,neoplastic,metabolic,etc.)or disorders of neuronal development and migration(Table3), and other situations presumed to be symptomatic but of unknown etiology or cryptogenous.Re-mote symptomatic seizure etiology seems to be one of the most important predictors of seizure intractability(Sillanpa¨a¨,1993). Neuropathology of refractory epilepsy mainly derives from surgical resected brain tissue.How-ever,the exact causative role of the lesion cannot be always established,mainly in cases of dual pathology.The risk for intractability varies with the different conditions(Vinters et al.,1993). Intractable focal epilepsy is often associated with structural lesions of the cerebral cortex,par-ticularly with cortical dysplasia(Awas et al.,1991; Fish et al.,1991;Palmini et al.,1995),in relation to the high and intrinsic epileptogenicity of these lesions(Palmini et al.,1995;Mattia et al.,1995) often involving an area more extended than seen on magnetic resonance imaging or in operation (Palmini et al.,1991;Andermann,1994).De-creases of GABAergic interneurons(Ferrer et al., 1992)or anomalies in synaptogenesis(Becker, 1991)have been reported.Hippocampal sclerosis is another cause of re-fractoriness and the most common pathological ﬁnding in temporal lobe epilepsy in surgical stud-ies(Mathern et al.,1995a,b,1996a).However, despite the large number of studies suggesting that hippocampal sclerosis leads to epilepsy,it is still possible that repeated seizures can produce or aggravate sclerosis(Swanson,1995).Whether early childhood seizures may be one cause of this particular hippocampal damage with a conse-focal epilepsy with complex partial seizures,bilat-eral temporal slow wave foci in the initial EEG and development of bilateral temporal slow wave foci during treatment seem also to constitute neg-ative prognostic factors(Runge,1996).。

贵州省金银花白粉病病原鉴定、生物学特性及防治药剂筛选

贵州省金银花白粉病病原鉴定、生物学特性及防治药剂筛选作者：崔文艳罗喜燕吴小云陈永超李明伟彭玲何朋杰来源：《南方农业学报》2023年第06期DOI：10.3969/j.issn.2095-1191.2023.06.012摘要：【目的】明確贵州省金银花白粉病的病原菌分类地位与生物学特性，筛选有效的防治药剂，为金银花白粉病的田间防治提供理论基础。

【方法】分别于贵州省铜仁市沿河县和遵义市绥阳县采集具有典型白粉病症状的金银花叶片和花蕾，对获得的菌株进行致病性测定，结合形态特征和ITS序列测序及系统发育进化分析对病原菌进行分类鉴定；采用孢子萌发法和喷施接种法分析温度对病原菌分生孢子萌发率和致病性的影响；结合室内毒力测定和温室药效试验评估6种常见杀菌剂对金银花白粉病的防控效果。

【结果】从贵州省遵义市和铜仁市金银花产区共获得33株疑似病原菌菌株，致病性测定结果表明，所有菌株均为金银花白粉病致病菌，其中代表性菌株BF1的形态特征与忍冬叉丝壳菌（Erysiphe lonicerae var. lonicerae）相似，且其ITS序列聚类分析显示与E. lonicerae var. lonicerae ITS序列聚为一类。

菌株BF1生物学特性测定结果显示，菌株BF1最适孢子萌发温度为24～26 ℃，最适致病温度为24 ℃。

防治药剂筛选试验结果显示，室内条件下1%多抗霉素、30%吡唑醚菌酯、5%已唑醇和0.3%四霉素对菌株BF1分生孢子萌发有较好的抑制作用，其EC50分别为0.3454、0.4347、0.5369和0.6315 g/L；温室条件下1%多抗霉素、30%吡唑醚菌酯、5%已唑醇和0.3%四霉素对金银花白粉病均表现出较好的防治效果，第2次药后的防治效果分别为88.53%、86.40%、84.00%和89.60%。

【结论】引起贵州省铜仁市和遵义市金银花白粉病的病原菌为忍冬叉丝壳菌。

0.3%四霉素水剂、1%多抗霉素水剂、30%吡唑醚菌酯悬浮剂和5%己唑醇乳剂能有效防控金银花白粉病，可在金银花生产中推广应用。

3302222a[1]

RESEARCH ARTICLEDelivery of GDNF by an E1,E3/E4deleted adenoviral vector and driven by a GFAP promoter prevents dopaminergic neuron degeneration in a rat model of Parkinson’s diseaseNA Do Thi 1,P Saillour 1,L Ferrero 2,JF Dedieu 2,J Mallet 1and T Paunio 1,31Laboratoire de Genetique Moleculaire de la Neurotransmission et des Processus Neurodegeneratifs,CNRS,Bat.CERVI,Hopital Pitie-Salpetriere,Paris,France;2Gencell SAS,Vitry sur Seine,France;and 3Department of Molecular Medicine,Biomedicum,Helsinki,FinlandA new adenoviral vector (Ad-GFAP-GDNF)(Ad-¼adenovirus,GFAP ¼glial ﬁbrillary acidic protein,GDNF ¼glial cell line-derived neurotrophic factor)was constructed in which (i)the E1,E3/E4regions of Ad5were deleted and (ii)the GDNF transgene is driven by the GFAP promoter.We veriﬁed,in vitro,that the recombinant GDNF was expressed in primary cultures of astrocytes.In vivo,the Ad-GFAP-GDNF was injected into the striatum of rats 1week before provoking striatal 6-OHDA lesion.After 1month,the striatal GDNF levels were 37pg/m g total protein.This quantity was at least 120-fold higher than in nontransduced striatum or after injection of the empty adenoviral vector.At 3months after viral injection,GDNF expression decreased,whereas the viral DNA remained unchanged.Furthermore,around 70%of the dopaminergic (DA)neurons were protected from degeneration up to 3months as compared to about 45%in the control groups.In addition,the ampheta-mine-induced rotational behavior was decreased.The results obtained in this study on DA neuron protection and rotational behavior are similar to those previously reported using vectors with viral promoters.In addition to these results,we established that a high level of GDNF was present in the striatum and that the period of GDNF expression was prolonged after injection of our adenoviral vector.Gene Therapy (2004)11,746–756.doi:10.1038/sj.gt.3302222Published online 15January 2004Keywords:GDNF;Parkinson’s disease;DA neurons;recombinant adenovirusIntroductionParkinson’s disease (PD)is a progressive neurodegen-erative disorder.It is characterized by tremor,bradyki-nesia,rigidity and postural instability that result from a loss of dopaminergic (DA)neurons of the nigrostriatal pathway.The best current therapy of PD is the administration of L -Dopa.However,L -Dopa loses its effectiveness as the disease progresses.Different ther-apeutic approaches are therefore being investigated such as the use of neurotrophic factors,1–4cell/tissue transplantation 5–7and gene transfer of trophic factors using recombinant adenovirus,recombinant adeno-associated virus (AAV)or lentivirus.8,9The ultimate goal of these approaches would be to arrest or to slow down the progressive degenerative process of the disease.Previous research in our laboratory 10,11and in others 12revealed that the progressive degeneration of DA neurons in an adult rat model of PD,13using an E1/E3defective adenovirus (ﬁrst-generation virus)encoding glial cell line-derived neurotrophic factor (GDNF),under a viral promoter was reduced.The effects obtained in vivo depend on the time and site of administration of recombinant virus.14,15Although these results were encouraging for gene therapy,the ﬁrst-generation ade-novirus had a toxic effect for transduced cells by inducing the synthesis of viral proteins within these cells.These proteins elicit an immune response in the injected brains,which generates toxicity.It is probable that the toxicity of the E1/E3defective virus is due to the E4region of the type 5adenovirus which is present in the recombinant virus.Yeh et al 16suggested that,in vivo ,a low level of E4expression could be cytotoxic to the recipient cells.In fact,Dedieu et al 17demonstrated that the E1,E3/E4defective adenovirus (third generation)was less toxic for transduced cells than the ‘ﬁrst-generation’E1/E3defec-tive virus.In addition,the infusion of E1,E3/E4defective virus elicited lower inﬂammatory responses,improved local tolerance and increased viral DNA persistence in the liver of a lacZ -transgenic mouse.Thus,an E1,E3/E4defective adenovirus represents progression on the path toward preclinical therapy.The aim of our work was to use the E1,E3/E4defective adenovirus to deliver GDNF,a therapeutic gene,into theReceived 17June 2003;accepted 29November 2003;published online 15January 2004Correspondence:J Mallet,Laboratoire de Genetique Moleculaire de la Neurotransmission et des Processus Neurodegeneratifs,Bat.CERVI,Hopital de la Pitie-Salpeˆtriere,83Bd.de l’Hopital,75013Paris,France Gene Therapy (2004)11,746–756&2004Nature Publishing Group All rights reserved 0969-7128/04$/gtbrain of a rat model of PD.The expression of GDNF was targeted to the astrocytes in the lesioned striatum. Astrocytes are the most abundant glial cells in the central nervous system(CNS),and are necessary for the survival of neurons in vivo18and in vitro.19These cells produce and secrete several growth factors,and among them,GDNF and cytokines.20–23In addition,following brain injury,the glialﬁbrillary acidic protein(GFAP) gene is upregulated in reactive astrocytes.24We therefore used the promoter of the GFAP gene,whose expression in the CNS is restricted to astrocytes,24,25to drive GDNF gene expression.Restriction of GDNF expression to a speciﬁc cell type limits the side effects caused by the expression of this gene in surrounding cells,thus facilitating the long-term expression of the transgene. Our results unequivocally showed that the recombinant Ad-GFAP-GDNF,in which the transgene was driven by a glial-speciﬁc promoter,prevented DA neurons death after striatal lesions induced by6-OHDA in the rats and improved the drug-induced rotational behavior.As compared to the E1/E3deleted adenovirus used previously in our laboratory,the cytotoxicity in injected animals was much lower.ResultsIn this work we constructed an E1,E3/E4defective recombinant adenovirus encoding rat GDNF under the control of a speciﬁc glial promoter,GFAP,by homo-logous recombination in Escherichia coli(see Materials and methods).This defective virus exhibits a large deletion in the E4region,which abrogates the synthesis of all E4-encoded gene products.17The virus was generated in the IGRP2cell line that transcomplements the E4viral function.16In vitro experimentsThe ability of the recombinant Ad-GFAP-GDNF to express GDNF wasﬁrst tested in primary cultures of astrocytes.The neurotrophic effect of this secreted protein on the survival of DA neurons was performed on mesencephalic cultures.Synthesis of GDNF by various types of cells.To determine whether astrocytes transduced with Ad-GFAP-GDNF express recombinant GDNF,cultivated rat astro-cytes were infected with the recombinant virus as well as an empty control at different doses.Conditioned medium(CM)and cellular pellets were collected at4,6 and8days after infection for analysis by ELISA.The quantity of endogenous GDNF secreted by noninfected astrocytes,or those infected with empty adenovirus was low(120716pg/ml)at all time points tested in CM.The amount of endogenous GDNF was close to the detection limit of the assay(20pg/ml)in the cellular pellet.In astrocytes infected with50viral particles(vp)/cell of Ad-GFAP-GDNF,0.370.05ng/ml of GDNF was secreted in the CM per day.GDNF levels of0.270.04, 0.470.07and0.370.08ng/ml were detected in the cellular pellet at days4,6and8,respectively. However,when astrocytes were infected with Ad-GFAP-GDNF at higher doses(500and103vp/cell), 50–60ng/ml of GDNF was secreted in the CM by105cells per day(Figure1)(P o0.0001for500vp;P¼0.0008for103vp as compared to control).At a dose of5Â103vp/cell,70ng/ml of GDNF was secreted in theCM from day4to day6,and it declined thereafter (Figure1)(P o0.0001as compared to control).In cellular pellets,about4074ng/ml of GDNF was found at thesethree doses at all time points tested.From this result,twodoses of virus,500and5Â103vp/cell,were chosen toinfect the mesencephalic cells.Mesencephalic cells infected with500and5Â103vp/cell of Ad-GFAP-GDNF secreted3079and80720pg/ml of GDNF in the the CM,respectively.In control cultures(cells noninfected by the virus),about35pg/mlof GDNF was found in the CM.In cellular pellets,0.570.1and0.870.12ng/ml ofGDNF were measured in cells infected with500and5Â103vp/cell,respectively,6days after infection.In cellular pellets of control cultures,0.670.09ng/ml ofGDNF was found.These results indicate that recombi-nant GDNF was not effectively synthesized by the mesencephalic cells infected with Ad-GFAP-GDNF.Survival of DA neurons.To test the effect of GDNF onthe survival of neuronal cells,104mesencephalic cellswere plated on a layer of nontransduced astrocytes, astrocytes infected with103vp/cell of Ad-GFAP-GDNFor onto collagen-coated coverslips(control).When plated on transduced astrocytes,457770 tyrosine hydroxylase(TH)-positive neurons were found.This was two-fold lower if plated on noninfected astrocytes(233723).The number of surviving TH-positive neurons was lowest(114718)on collagen-coated coverslips(Figure2).Figure1GDNF levels in cultured astrocytes infected with recombinantvirus.Primary astrocytes were infected with Ad-GFAP-GDNF at differentdoses.d1:500vp/cell;d2:103vp/cell;d3:5Â103vp/cell;C:control, noninfected astrocytes.In all,50–60ng/ml of GDNF was released by105cells per day with both doses d1and d2.At a higher dose(d3),about70ng/ml of GDNF was released by105cells/day until day6after infection.Thenthe quantity of GDNF decreased at day8.*P o0.0001d1versus control atthree times analyzed;**P¼0.008,0.0004,o0.0001d2versus control atthree times analyzed,respectively;***P o0.0001,o0.0001,0.0002d3versus control at three times analyzed,respectively.Degeneration of DA neurons prevented by Ad-GDNFNA Do Thi et al747Gene TherapyIn vivo experimentsThe effect of striatal overexpression of GDNF on the DA neuron survival and motor function in a rat model of PD was investigated by direct in vivo delivery of the transgene,using recombinant Ad-GFAP-GDNF.Body weight.Injection of large doses of recombinant GDNF protein has been found to cause loss of body weight in rats.26No signiﬁcant differences in weight were observed among the treatment groups over the entire period of experimentation.Thus the quantity of transgenic GDNF detected in the striatum did not affect the body weight of rats.GDNF expression in intact animals.In preliminary experiments ,different doses of virus (107,108,5Â108,109and 3Â109vp/rat)were used to determine an optimal dose both in terms of level of GDNF expression and inﬂammatory reaction.GDNF expression was assessed by ELISA in striatum obtained from nonlesioned animals that were killed 10days,4,6and 12weeks after vectorinjection.As shown in Table 1,at doses of 108and 5Â108vp/rat,the striatal GDNF content was the highest at all times analyzed as compared to other doses.At a lower dose of the virus (107vp/rat),GDNF levels were 10-fold lower than those measured in rats that had received 108vp of virus,at all time points studied.At doses of 109and 3Â109vp/rat,12–20pg/m g GDNF protein was found (Table 1)in transduced striatum,but a marked,strong inﬂammatory reaction was observed (data not shown).However,the GDNF protein levels decreased with time at all doses used (Table 1).Analysis with semiquantitative competitive polymer-ase chain reaction (sqc-PCR)showed that the level of viral DNA in injected striatum (108vp/rat)did not change between 10days and 12weeks after viral administration (Figure 3).From these results,we used 108vp/rat in the following experiments.GDNF expression in lesioned rats.The efﬁcacy of adenovirus-mediated GDNF gene transfer was tested on a rat model of PD.13Adult rats were injected stereo-taxically with 108vp of Ad-GFAP-GDNF (G group)into the left striatum as described in Materials and methods.At 1week after viral injection,rats were anesthetized and received stereotaxic injection of 6-OHDA.Striatum and substantia nigra (SN)were dissected out of animals killed at 4,6and 12weeks after viral injection and the GDNF levels in these tissues were determined by ELISA.As shown in Table 2,the GDNF protein levels were 37–40times higher (37.3and 41pg/m g corresponding to 70and 75ng of GDNF per striatum,respectively)in Ad-GFAP-GDNF-transduced striatum at 4and 6weeks as compared to both control groups (OHDA ¼OH group;empty ¼E group),as well as to the noninjected side.The GDNF quantity was decreased at 12weeks (17.2pg/m g corresponds to 35ng of GDNF per striatum)afterviralFigure 2Survival of mesencephalic cells in culture.The survival TH (þ)neurons was two-fold higher on transduced astrocytes (G)than on noninfected cells (A,P ¼0.002)and four-fold higher as compared to control (C,P ¼0.0003).C:neurons on collagen-coated coverslips used as control;A:neurons on noninfected astrocytes;G:neurons on astrocytes infected with Ad-GFAP-GDNF.Table 1GDNF protein levels measured from transduced striatum of intact animals after Ad-GFAP-GDNF injection Doses 107vp 108vp 5Â108vp 109vp 3Â109vp 10days 3.25723579327814.67215734weeks 1.470.227.87223.47215.37316746weeks 2.570.7277834.77522.27220.147212weeks1.370.1214.67626.37312.5731273GDNF protein levels (pg/m g total protein),measured from injected striatum of intact animals (three animals per point),were high at doses of 108and 5Â108vp/rat as compared to other doses of virus.At 12weeks after viral treatment,the quantity of GDNF decreased with all doses used.Values are means 7s.e.m.Figure 3Viral DNA levels of injected striatum,in nonlesioned rats.The relative viral DNA amount in rats injected with 108vp/rat of Ad-GFAP-GDNF was unchanged from day 10to week 12(three animals per point).P ¼0.5,4weeks versus 10days;P ¼0.9,6weeks versus 10days;P ¼0.9,12weeks versus 10days.Degeneration of DA neurons prevented by Ad-GDNFNA Do Thi et al748Gene Therapyinjection.To explain the decline of GDNF levels in Ad-GFAP-GDNF-injected striatum at a later stage,sqc-PCR was performed to determine the relative quantity of the virus at different times after viral injection.As shown in Figure 4,the viral DNA levels were unchanged during 12weeks of experiment,which suggests a downregulation of GFAP promoter in vivo rather than a loss of injected viral DNA.In the SN,the GDNF protein levels were similar in the injected side as compared to the noninjected side and in all groups of animals (Table 2).The viral DNA levels were not detectable in the SN by sqc-PCR.Amphetamine-induced rotation test.The impact of GDNF overexpression on the behavior of the animals was assessed during 12weeks after viral vector injection.As early as 2weeks after the striatal 6-OHDA lesion (3weeks after viral injection),rats injected with Ad-GFAP-GDNF into the striatum began to display reduced amphetamine-induced rotations.As shown in Figure 5,3,4,6and 12weeks after Ad-GFAP-GDNF treatment,rats exhibited a signiﬁcant reduction inTable 2GDNF protein levels from striatum and SN of lesioned animalsStriatumSubstantia nigraInjected sideNoninjected sideInjected sideNoninjected sideAd-GFAP-GDNF (G group)4weeks *#137.375.10.2970.070.1570.050.2170.096weeks *#14176.50.3470.130.270.060.2370.0612weeks *#117.274.20.2670.060.2670.040.2270.05Empty virus (E group)4weeks 0.1470.020.0970.010.270.040.1570.036weeks 0.0870.010.0770.0030.1270.020.270.0512weeks0.0970.040.0770.0020.1370.020.1470.056-OHDA alone (OH group)4weeks 0.1270.030.1670.030.1670.020.1470.016weeks 0.1470.020.1570.060.1770.020.1370.0112weeks0.1670.010.1270.020.270.050.270.01GDNF protein levels (pg/m g total protein)from striatum and SN measured by ELISA in lesioned animals treated with Ad-GFAP-GDNF (108vp/rat),with empty adenovirus (108vp/rat)or in naive animals that did not receive treatment before inducing 6-OHDA lesion.Seven animals were used per point.GDNF protein levels decreased with time in transduced striatum (G group)and remained unchanged in the injected striatum of E and OH groups.The GDNF levels were similar in the SN (injected and noninjected side)of all groups.Values are means 7s.e.m.*P o 0.0001different from noninjected side;#P o 0.0001G versus E group;P o 0.0001G versus OHgroup.Figure 4Viral DNA levels of injected striatum in lesioned rats at 4,6and 12weeks after viral treatment.The relative quantity of viral DNA was similar during our experiment from 4to 12weeks (ﬁve animals per point).P ¼0.9,6weeks versus 4weeks;P ¼0.6,12weeks versus 4weeks.Figure 5Motor performance of the animals using the drug-induced rotation test.Rats were injected with D -amphetamine (2.5mg/kg,i.p.)and their behavior was recorded for 90min.At 3,4,6and 12weeks after viral injection,rats injected with Ad-GFAP-GDNF (G group)exhibited a signiﬁcant reduction in ipsilateral rotational behavior compared with control groups (OH and E groups).P ¼0.008,G versus OH group (3weeks);P ¼0.2,G versus E group (3weeks);P o 0.0001,G versus E and OH groups (4weeks);P o 0.0001,G versus E and OH group (6weeks);P ¼0.006,G versus E group (12weeks);P ¼0.0002,G versus OH group (12weeks).Degeneration of DA neurons prevented by Ad-GDNF NA Do Thi et al749Gene Therapyipsilateral rotational behavior compared with control groups(OH and E groups)(P o0.0001).In rats injected with empty virus,the rotation score was not signiﬁcantly different from that of the animals that received6-OHDA alone at3,4and6weeks after viral injection(P¼0.08,0.4and0.3,respectively).Protection of DA neurons in the SN.Survival of DA neurons was analyzed throughout the SN as described in Materials and parison of the percentage of the TH-positive cells in the SN(average results from the three levels analyzed)revealed that about70%of DA neurons remained in the G group as compared to about 45%in the control groups at all three times examined (P o0.0001,0.01and0.0004,4,6and12weeks, respectively)(Figures6,7a and b).This result suggested that a signiﬁcant protection of DA neurons was found in animals treated with Ad-GFAP-GDNF.Immunoreactivity in the striatum.Following an injec-tion of6-OHDA into the striatum,there is an immediate toxic damage to the DAﬁbers and axons followed by a rapid degeneration of their terminals.4,27We used NeuN staining to localize the lesion in the striatum(Figure9d), and immunohistochemistry for the TH to assess the extent of denervation induced by intrastriatal6-OHDA lesions(Figure8a and b).The extent of denervation was prominent in the central and dorsal parts of the injected striatum in all animals analyzed.The intensity of TH-positiveﬁber staining,measured by optical density,in the injected striatum(average results from the three levels analyzed)was similar in both E and OH groups (Figure8b).It was reduced by about70–75%(P o0.0001) in the injected side versus the noninjected side at4weeks and by about80–85%(P o0.0001)at6and12weeks. The TH intensity was slightly higher in animals of G group(þ7%)at4,6and12weeks as compared to controls,but it did not reach statistical signiﬁcance (P¼0.2)(Figure8a).Abundant TH immunoreactive proﬁles(dots)of different sizes(Figure8c and f)were observed in the lesion sites of the striatum.Some of these patterns were scattered throughout the parenchyma in animals of G, OH and E groups.However,in animals treated with Ad-GFAP-GDNF(Figure8c),the number of these TH immunoreactive proﬁles was increased as compared to control animals at all three times analyzed(Figure8f).At higher magniﬁcation,we observed TH-positiveﬁbers with numerous axonal varicosities which displayed different intensity of TH staining(Figure8d,compared with Figure8g(control)).In globus pallidus,we observed the TH immunoreactive area,which appears to correspond to the axonal sprouting of TH-positive ﬁbers in animals treated with Ad-GFAP-GDNF vectors (Figure7c).The TH-positiveﬁbers were also observed in the entopeduncular nucleus of these rats(Figure7d). These patterns were not seen in any of the animals of control groups.The GDNF transgene expression in transduced stria-tum was visualized by anti-GDNF antibody.As shown in Figure9a and b,the striatal astrocytes of animals treated with Ad-GFAP-GDNF were stained with GDNF antibody.We also determined the effect of the viruses on the size of the striatum by analyzing the surface of10sections per brain between the coordinates APþ1.7and APþ0.2. The injected striatal size was not modiﬁed in G(P¼0.8), E(P¼0.7)and OH(P¼0.4)groups at4weeks as compared to the noninjected side.However,we observed a nonsigniﬁcant atrophy,7%as compared to controlate-ral size,of the injected striatum of G(P¼0.5)andEFigure6Rescue of TH immunoreactive neurons in the SN.Signiﬁcantincrease in the percentage of TH immunoreactive neurons was observed inlesioned rats treated with Ad-GFAP-GDNF(G group)compared with ratsinjected with empty adenovirus(E group)or with lesioned rats(OHgroup).P o0.0001,G versus E group(4weeks);P¼0.001,G versus OHgroup(4weeks);P¼0.01,G versus E group(6weeks);P¼0.003,Gversus OH group(6weeks);P¼0.0004,G versus E group(12weeks);P o0.0001,G versus OH group(12weeks).Figure7TH staining of injected brain.Many TH-positive neuronsremained in the rostral,middle and caudal parts of the SN in animalstreated with Ad-GFAP-GDNF(a),while fewer cells survived in animalslesioned by6-OHDA(b),12weeks after treatment.The presence of TH-positiveﬁbers(asterisk)was seen in globus pallidus(c)and inentopeduncular nucleus(d),4weeks following Ad-GFAP-GDNF injection(injected side¼left side,arrow;right side¼intact side).Scale barrepresents(a,b)250m m and(c,d)150mm.Degeneration of DA neurons prevented by Ad-GDNFNA Do Thi et al750Gene Therapy(P ¼0.6)groups at 6and 12weeks.In the OH group,very mild atrophy was seen (4%as compared to the controlateral side)at 12weeks,but not signiﬁcant as assessed by one-way analysis of variance (ANOVA),P ¼0.6.Inﬂammatory response.Injuries to the brain result in a rapid inﬂammatory reponse that typically involves recruitment and inﬁltration of different cell populations.Immunohistochemistry using CD4and CD8antibodies allows one to determine the localization of reactive lymphocytes.CD4immunoreactive cells were most numerous at the injection sites of the adenoviral vector with or without transgene,and at the 6-OHDA lesion in all treatment groups (Figure 9e).They were also scatteredthroughout the parenchyma and close to the blood vessels.A few CD8immunoreactive cells were particu-larly concentrated at the injection sites of the adenoviral vector and around the 6-OHDA lesion (Figure 9f).We did not observe more inﬂammation in G and E groups as compared to the OH group,with both CD4and CD8antibodies (Table 3).Astrocytic response to injury was assessed by using an antibody against GFAP .Glial ﬁbrillaly acidic protein,an intermediate ﬁlament protein,is expressed abundantly in astrocytes during development 28of the CNS and in reactive astrocytes (astrogliosis)following CNS in-jury.24,25Reactive astrocytes,characterized by a signiﬁ-cant increase in the GFAP intensity,cellular hypertrophy and increase in the density of GFAPimmunoreactiveFigure 8TH immunostaining of the striatum,4weeks after viral treatment.On the intact side,the TH staining intensity was high throughout the striatum (a,b,right side).After intrastriatal lesion,the TH staining was almost lost at the site of 6-OHDA injection (b,left side).By contrast,Ad-GFAP-GDNF-treated animals had a more lasting TH intensity on the ipsilateral side (a,left side).High-power magniﬁcation of boxed area in (b)showed that the axonal terminals in the striatum were degenerated at the lesion site (e,asterisk),whereas some spared terminals remained (arrow).In these GDNF-treated animals,numerous TH-positive axonal proﬁles (c,dots)and TH immunoreactive ﬁbers with varicosities and sprouting (d,arrow)were seen in the denervated striatum.In the striatum of 6-OHDA lesioned animals,the TH-positive proﬁles (f)and TH immunoreactive ﬁbers with varicosities (g)were less numerous.Scale bar represents (a,b)200m m and (c–g)50mm.Figure 9Immunostaining of transduced striatum,4weeks after Ad-GFAP-GDNF treatment.At the injected site,a halo of GDNF was seen with GDNF-positive astrocytes (a);at low magniﬁcation astrocytes stained with GDNF antibody (b).Numerous cells stained with CD4(e)and CD8(f)at the injected site.In (d)the site of 6-OHDA lesion was stained by NeuN antibody.Inside the lesion,the neurons were degenerated,while around the lesion the nuclei of neurons were stained by NeuN.Reactive astrocytes were stained by GFAP antibody (c)at the injected site of the striatum.Scale bar represents (a)35m m,(b)200m m,(c)50m m,(d)200m m and (e,f)100mm.Degeneration of DA neurons prevented by Ad-GDNF NA Do Thi et al751Gene Therapyprocesses,were detected throughout the ipsilateral striatum.The GFAP staining was particularly intense at the lesion with a dense network of cell bodies and processes in all study groups (Figure 9c).DiscussionThe aim of the present study was to assess the ability of GDNF,expressed by an improved E1,E3/E4defective recombinant adenovirus in which the GDNF gene is driven by a glial-speciﬁc promoter,to preserve the integrity of the nigrostriatal DA system (cell bodies,axonal terminals)and the normal motor function after administration of the virus into the striatum before inducing 6-OHDA lesion.Our interest was also to test the GFAP promoter for PD therapy since this promoter was described to direct speciﬁcally transgene expression in astrocytes.24,25In our cell cultures,GDNF protein was not synthe-sized by the mesencephalic cells infected with the recombinant GFAP-GDNF adenovirus,whereas this trophic factor was produced and secreted by the transduced astrocytes (Figure 1).Morelli et al 29observed that only cultured neocortical neurons,infected with a recombinant defective adenovirus vector encoding FasL under the control of the neuronal-speciﬁc promoter NSE (RAd-NSE-FasL),released the cytotoxic Fas ligand into the culture supernatant.Neurons transduced with a vector under the control of a glial-speciﬁc promoter (RAd-GFAP-FasL)were unable to release the FasL cytotoxic activity.Thus,the expression of the transgene was cell-type restricted when the transcription was directed from a glial-or a neuronal-speciﬁc promoter in the adenoviral vector.In vivo ,in a rat model of PD,immunohistochemical experiments,performed in the transduced striatum,demonstrated that the expression of the transgene (GDNF)was conﬁned to astrocytes (Figure 9a and b).This observation was supported by the results obtained from ELISA tests (Table 2).After an intrastriatal injection of Ad-GFAP-GDNF,the GDNF protein levels were high in transduced striatum (37–41pg/m g protein from 4to 6weeks;Table 2).At 12weeks the quantity of GDNF protein decreased,whereas the levels of adenoviral DNAremained unchanged from weeks 4to 12(Figure 4).The decline of the transgene expression could result from the host immune responses to the vector in infected cells 30,31or due to the down regulation of the promoter.12In our study,the decline in the GDNF expression was unequi-vocally the result of a downregulation of the GFAP promoter rather than the loss of adenovirus-infected cells,since the quantity of viral DNA in the transduced striatum did not change during the experiment (Figure 4).Using the RSV promoter to drive the expression of the GDNF,Choi-Lundberg et al 32found that GDNF protein and GDNF DNA levels decreased simultaneously from weeks 1to 7.In our study,although the GDNF protein level decreased,it remained relatively high at 12weeks (17pg/m g protein;Table 2).Armentano et al 33and Dedieu et al 17showed that the deleted E1,E3/E4recombinant adenoviruses were unable to sustain a strong and stable transgene expression when under the control of the CMV and RSV promoters.Thus the long-lasting presence of the recombinant GDNF in our experiment cannot be attributed solely to the E1,E3/E4Ad-GFAP-GDNF backbone.The prolongation of the GDNF expression we obtained was probably the consequence of the activity of the GFAP promoter.Despite a downregulation of the GFAP promoter at 12weeks,its remaining activity would still be sufﬁcient to induce the late and high-level expression of the transgene in the astrocytes.In four animals we even observed GDNF expression in the transduced striatum 5months after Ad-GFAP-GDNF injection (unpublished results).In addition,the recombinant E1,E3/E4defective adenovirus used in the present study appeared to be weakly immunogenic in the brain.We did not observe an increased inﬂammation in the lesioned brains after GFAP-GDNF or empty virus injection as compared to the OHDA-injected animals (Table 3).Moreover,the transduced striatum sizes were not reduced.These results suggest that this is an improvement of the adenoviral vector compared to the ﬁrst-generation adenovirus used previously by our group.11Another important result was that approximately 70%(as compared to about 45%in the controls)of the nigral DA neurons were still present at 12weeks when the recombinant Ad-GFAP-GDNF had been injected into the striatum,1week before inducing the intrastriatal 6-OHDA lesion.The ratios of protected neurons did not change with time from week 4to week 12after the viral treatment (Figure 6).As the protection of the DA neurons was not complete,it is possible that the recombinant GDNF,synthesized by striatal transduced astrocytes and transported by retrograde axonal transport,was not sufﬁcient in the SN (Table 2).The results obtained in this work are consistent with previous studies by our group 11and others 9,14using Ad/AAV-GDNF injected in the striatum.In addition,these authors 9,11,14found (1)that the intensity of the TH immunoreactivity was increased in the injected striatum,as compared to control 9,11,14and (2)that the axonal sprouting was present in the striatum and the globus pallidus.9In our rats treated with Ad-GFAP-GDNF,although the sprouting was observed in the striatum and the globus pallidus (Figures 7and 8),the intensity of the striatal TH staining was not modiﬁed in the injected side.This may be due to the lowTable 3Semiquantitative estimation of the inﬂammation in injected animals4weeks 6weeks 12weeks GE OH G E OH G E OH 7404040504242636353+524252424242424342++132414141223030212+++050404041303020203++++000000010000Number of animals from G,E and OH groups where the inﬂammation was produced in the brains from 4to 12weeks after treatment.For each animal,10CD8and CD4-stained sections were examined and scored as described in Materials and methods.First values in each column (G,E,OH)were estimated on CD8-stained sections,and second values (italic)were estimated on CD4-stainedsections.Degeneration of DA neurons prevented by Ad-GDNFNA Do Thi et al752Gene Therapy。

Effect of repeated ipsapirone treatment on hippocampal excitatory

Keywords: 5-HT Ž5-hydroxytryptamine, serotonin.; Ipsapirone; WAY-100635; p-Chlorophenylalanine; Hippocampus; Excitatory synaptic transmission; 5-HT1A receptor; Excitatory postsynaptic potential; Ultrasonic vocalization; Anxiolytic
c Institute of Neurobiology, Troponwerke GmbH & Co. KG, Berliner Strasse 156, D-51063 Cologne, Germ1996; accepted 31 December 1996
1. Introduction
The pyrimidinylpiperazine anxiolytics such as buspirone, gepirone, ipsapirone and tandospirone have marked acute inhibitory effects on neuronal transmission in the rat hippocampus. In vitro they share the ability to hyperpolarize rat hippocampal pyramidal cells by the activation of 5-HT1A receptor G-protein-linked Kq channels ŽColino and Halliwell, 1987; Andrade and Nicoll, 1987.. 5-HT1A receptors may also be located on glutamatergic terminals, thereby contributing to the depressant effects on excitatory transmission in the hippocampus ŽSchmitz et al., 1995.. The decrease in spontaneous firing rate of hippocampal

cx3cl1 cx3cr1-mediated microglia activation plays a detrimental role in ischemic mice brain via

ORIGINAL ARTICLECX3CL1/CX3CR1-mediated microglia activation plays a detrimental role in ischemic mice brain via p38MAPK/PKC pathwayYong Liu 1,5,Xiao-Mei Wu 2,3,5,Qian-Qian Luo 2,4,Suna Huang 4,Qing-Wu Qian Yang 1,Fa-Xiang Wang 1,Ya Ke 3and Zhong-Ming Qian 2,4INTRODUCTIONMicroglia is key modulators of the immune response in the brain.1Under normal physiologic conditions,these cells constantly survey their microenvironment for noxious agents and injurious processes,2respond to extracellular signals and are responsible for clearing debris and toxic substances by phagocytosis,thereby maintaining normal cellular homeostasis in the central nervous system.3Under the pathologic circumstance,these cells could be activated at very early stage.4In animal models of cerebral ischemia,the processes of microglial activation have been studied extensively,however,the exact function of these activated cells is not fully understood 5and the ﬁndings reported are controversial.6–8The answers to the question of whether activated microglial responses are destructive or bene ﬁcial after ischemic injury remains equivocal.4,8Microglial activation is usually regulated by the chemokine fractalkine (CX3CL1)CX3CL1and its receptor,CX3CR1.4CX3CL1is a relatively new member of the chemokine (chemotactic cytokine)family and the sole member of the CX3C chemokine class,9–10which exists in both membrane bound and soluble forms.9In contrast to many other chemokines,CX3CL1binds only one receptor CX3CR1.11In the brain,CX3CL1is a unique chemokine,being constitutively expressed by neurons where it is tethered to the extracellular membrane by a mucin stalk.9,12The CX3CL1receptor CX3CR1is a G-protein-coupled receptor and exclusively expressed by microglia.13The CX3CL1/CX3CR1signaling pathway has been shown to play an important role in the maintenance of neural –immune communication and the bidirectional interaction between neurons and microglia in health and disease.14,15A number of studies have been conducted to investigate the role of CX3CL1/CX3CR1signaling in brain ischemic injury,however,the relevant ﬁndings are also controversial.The destructive or bene ﬁcial roles of CX3CL1/CX3CR1in brain ischemic injury have also been reported.Mice de ﬁcient in CX3CL1were found to be less susceptible to cerebral ischemia –reperfusion injury when compared with wild-type littermates.16And lack or de ﬁciency of CX3CR1was shown to reduce signi ﬁcantly ischemic damage and in ﬂammation in mice with focal cerebral ischemia 171Department of Neurology,Xinqiao Hospital,The Third Military Medical University,Chongqing,China;2Department of Neurobiology,Institute for Nautical Medicine,Nantong University,Nantong,China;3School of Biomedical Sciences,Faculty of Medicine,The Chinese University of Hong Kong,New Territories,Hong Kong,China and 4Laboratory of Neuropharmacology,Fudan University School of Pharmacy,Pu Dong,Shanghai,China.Correspondence:Dr Y Ke,School of Biomedical Sciences,Faculty of Medicine,The Chinese University of Hong Kong,New Territories,Hong Kong,China or Dr ZM Qian,Laboratory of Neuropharmacology,Fudan University School of Pharmacy,Pu Dong,Shanghai 201203,China.E-mail:yake@.hk or qianzhongming@The studies in our laboratories were supported by the Competitive Earmarked Grants of The Hong Kong Research Grants Council (GRF 466713),National 973Programs (2011CB510004and 2014CB541604),General Grant of National Natural Science Foundation of China (NSFC;81070930,81471108,31271132and 31371092),and Key Project Grant of NSFC (31330035-2013).5These authors shared ﬁrst authorship.Received 23February 2014;revised 8April 2015;accepted 13April 2015Journal of Cerebral Blood Flow &Metabolism (2015),1–9©2015ISCBFM All rights reserved 0271-678X/15$and to suppress activation and neurotoxicity of microglia/macro-phage in experimental ischemic stroke.18In addition,CX3CL1-and CX3CR1-knockout mice revealed to have less severe brain injury on permanent19and transient20middle cerebral artery occlusion. These studies suggested that CX3CL1–CX3CR1expression is detrimental to recovery after ischemic injury.15,16However,it has also been reported that CX3CR1deﬁciency worsens the behavioral impairment induced by transient global cerebral ischemic injury and silencing CX3CR1expression exacerbates the learning and memory deﬁcits.4Moreover,administration of exogenous CX3CL1 was found to reduce ischemia-induced cerebral infarct size and neurologic deﬁcits in in vivo murine models of permanent middle cerebral artery occlusion.19These conﬂicting data to date do not provide a coherent conclusion on the role of CX3CL1/CX3CR1in brain injury and disease.4,20To further explore the roles of this signaling pathway andmicroglial activation in brain ischemic injury,we investigated theeffects of CX3CR1siRNA(silencing CX3CR1expression)onexpression of CX3CR1,p38mitogen-activated protein kinase(p38MAPK),protein kinase C(PKC),tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,and IL-6,and also microglia activation,white matter lesions,and cognitive function in the brain in mice modelof bilateral common carotid artery stenosis(BCAS)in vivo.We alsoexamined the effects of the addition of exogenous CX3CL1withCX3CR1siRNA and SB2035080(SB,a p38MAPK inhibitor)on theexpression of TNF-αand IL-1βin OGD(oxygen–glucose depriva-tion)treated BV2microglial cells in vitro.The p38MAPK waschosen to be examined because this signaling molecule has beenwell documented to be associated with the expression ofinﬂammatory and cytotoxic mediators.21Ourﬁndings indicatedthat CX3CL1,acting on the CX3CR1of microglia,activates p38MAPK/PKC and promotes the generation of TNF-αand IL-1βinducing a detrimental effect in the brain of ischemic mice under our experimental conditions.MATERIALS AND METHODSMaterialsUnless otherwise stated,all chemicals were obtained from Sigma Chemical Company,St.Louis,MO,USA.Rabbit polyclonal TNF-αwas purchased from Afﬁniti Research,Devon,UK,and rabbit monoclonal IL-1βand rabbit polyclonal IL-6from Lab Vision Corporation,Fremont,CA,USA. Rabbit polyclonal anti-CX3CR1was obtained from Merck Millipore,Billerica, MA,USA,antibodies against p38MAPK and PKC were purchased from CST,Cell Signaling Technology,Danvers,MA,USA and TNF-αand IL-1βELISA(enzyme-linked immunosorbent assay)kits from R&D Systems China,Shanghai,PRC.Sheep antirat biotinylated IgG,antirabbit secondary antibody–conjugated horseradish peroxidase(HRP),and HRP–streptavidin were obtained from Zhongshan Biotech,Beijing,China;rat monoclonal anti-CD11b antibody from AbD Serotec,Kidlington,UK;and Bradford assay kit from Bio-Rad,Hercules,CA,USA.CX3CR1siRNA(against CX3CR1 retrovirus)and negative siRNA(negative control)were obtained from GeneChem,Shanghai,PRC.Animals,BV2Microglia,and Primary Cultured NeuronsMale C57BL/6J mice(8to10weeks)weighing20to24g were supplied by the Animal Center of the Third Military Medical University(Chongqing, China)and housed in individual cages under a standard12-hour light–dark cycle with water and food supplied ad libitum.All animal handling and surgical procedures were approved by the Animal Research Ethics Committee of the Third Military Medical University in accordance with the guidelines of the Chongqing City Health Department on Animal Care. The experiments were performed in accordance with the Animal Research: Reporting In Vivo Experiments guidelines.BV2microglial cells were cultured in Dulbecco's modiﬁed Eagle's medium(DMEM;Gibco,Carlsbad,CA,USA)supplemented with10%fetal bovine serum(Gibco)and100μg/mL penicillin–streptomycin(Gibco)at 37°C in humidiﬁed atmosphere containing5%CO2.22The primary cortical neuronal culture was prepared from embryonic day14to15C57BL/6J mice using a method as described previously.23Experimental designIn vivo study.Two-vessel occlusion caused tissue injury in C57BL/6mice, which is highly variable because of the anatomic variations of the posterior communicating artery.24–26We therefore premeasured cerebral bloodﬂowbefore and2hours after BCAS with laser-Dopplerﬂowmetry as describedpreviously27to exclude animals that are less susceptible(cerebral blood ﬂow2hours after BCAS/cerebral bloodﬂow before⩾65%)to ischemic insult.To determine the role of CX3CL1/CX3CR1-mediated activation ofmicroglia and the relevant mechanisms under the conditions of ischemia,a total of27susceptible C57BL/6J mice were randomly assigned into three groups.The mice in BCAS or BCAS+siRNA group were pretreated with3μL of saline(BCAS group,n=9)or CX3CR1siRNA(BCAS+siRNA group,n=11) by intracerebroventricular injection,andﬁve days later,underwent with BCAS.The animals in the sham-operated group(n=7)underwent the same surgical procedure as the above two groups without BCAS.At day14after BCAS,the expression of CX3CR1,p38MAPK,and PKC,and the contents of TNF-α,IL-1β,and IL-6were determined by Western blot assay,and also microglia activation and white matter lesions were evaluated by histochemical method,cognitive function assessed by water maze test, and the expression of CX3CR1was also assessed by immunostain analysis.In vitro study.To further explore the relevant mechanisms involved in the role of fractalkine/CX3CR1-mediated activation of microglia under ischemia,the BV2microglial cells were treated with Oxygen–glucose deprivation(OGD)in the presence of CX3CL1(0,12.5,25,and50ng/mL),or infected with10Multiplicity of Infection(MOI)CX3CR1siRNA or negative siRNA for72hours or pretreated with20μmol/L of SB2035080only for 1hour,before subjected to OGD for4hours in the presence of0or50ng/ mL of CX3CL1.To investigate whether neurons prepared from C57BL/6J mice could secrete and express CX3CL1and whether OGD affect the release and expression of CX3CL1in neurons,neurons were treated with OGD for4hours and then reperfusion(R)for24hours.The contents of CX3CL1in neuronal medium was detected by ELISA and the expression of CX3CL1in neurons were observed by immunostaining.OGD was performed by exposing the cells to serum-free DMEM without glucose in a hypoxic incubator(INVIVO2,RUSKINN,Sanford,Maine,USA)with1%O2, 94%N2,and5%CO2at37°C for4hours as previously described.28After the treatments,the measurements of TNF-α,IL-1β,and CX3CL1were conducted using ELISA.Intracerebroventricular InjectionInjections(intracerebroventricular)were accomplished via an indwelling guide cannula stereotaxically implanted into the right lateral cerebral ventricle(bregma−0.58mm,lateral1.20mm,and depth2.25mm).28Bilateral Common Carotid Artery StenosisMale C57BL/6J mice were subjected to BCAS,which was performed by applying the microcoils(Sawane Spring,Osaka,Japan)with an inner diameter of0.18mm to common carotid arteries(CCAs).26Brieﬂy,both CCAs were exposed and freed from their sheaths,through a midline cervical incision,after the mice were intraperitoneally anesthetized with sodium pentobarbital(50mg/kg).Two4to0silk sutures were placed around the distal and proximal parts of the right CCA.Then,the artery was gently lifted by these sutures and placed between the loops of the microcoil just below the carotid bifurcation.The microcoil was twined by rotating it around the CCA.After30minutes,another microcoil of the same size was twined around the left CCA.The rectal temperature was maintained between36.5°C and37.5°C.Sham-operated mice underwent the same surgical procedure without using coils.Western Blot AssayThe expression of CX3CR1,p38MAPK,PKC,TNF-α,IL-1β,and IL-6in brain tissue of the mice were determined as described previously.29Total protein content was determined using the Bradford assay kit(Bio-Rad).The blots were probed with primary antibodies:rabbit polyclonal anti-CX3CR1 (1:1,000),p38MAPK(1:1,000),PKC(1:1,000),rabbit polyclonal TNF-α(1:500), rabbit monoclonal IL-1β(1:1,000),and rabbit polyclonal IL-6(1:1,000),and then antirabbit secondary antibody–conjugated horseradish peroxidase (1:2,500).The intensity of the speciﬁc bands was detected and analyzed by Odyssey infrared imaging system(Li-Cor Biosciences,Lincoln NE,USA).To ensure even loading of the samples,the same membrane was probed with rabbit antiratβ-actin polyclonal antibody at a1:2,000dilution.CX3CL1/CX3CR1and microglia activationY Liu et al 2Enzyme-Linked Immunosorbent AssayTNF-αand IL-1βconcentrations in the culture medium of BV2microglia and CX3CL1in the culture medium of neurons were determined using commercially available ELISA kits according to the manufacturer ’s instruction (R&D Systems,China).The optical density at 450nm was read by using an ELX-800microplate assay reader (Elx800,Bio-tek,Winooski,VT,USA).The average absorbance values for each set of standards and samples were calculated from the standard curve.30ImmunohistochemistryCoronal sections (30μm)were treated with 3%H 2O 2in 0.01mol/L phosphate-buffered saline and preincubated in 5%normal goat serum.Sections were then incubated with the primary antibody,rat monoclonal anti-CD11b antibody (1:100)at 4°C overnight,then with sheep antirat biotinylated IgG (1:200)for 1hour at room temperature and ﬁnally incubated in HRP –streptavidin (1:200)for 1hour at room temperature.The color reaction was conventionally developed with 3,3′-diaminobenzidine and H 2O 2.White Matter Lesion EvaluationAt day 14after BCAS,the brains were removed and post ﬁxed in 4%paraformaldehyde in 0.1mol/L phosphate buffer (PB)(pH 7.4)for 12hour,and then stored in 30%sucrose in 0.1mol/L PB (pH 7.4).Serial coronal sections were cut on a cryostat,spanning from the anterior aspect oftheFigure 1.CX3CR1siRNA signi ﬁcantly inhibits the increased expression of CX3CR1,p38MAPK,and PKC,and microglia activation induced by bilateral common carotid artery stenosis (BCAS)in mice brain in vivo .The mice in BCAS (n =9)or BCAS+siRNA (n =11)group were pretreated with saline or CX3CR1siRNA,and then underwent with BCAS.The animals in the sham-operated group (n =7)underwent the same surgical procedure as the above two groups without BCAS.At day 14after BCAS,the expression of CX3CR1,p38MAPK,and PKC (A and B )was determined by Western blot assay.The expression of CX3CR1was also assessed by immunostain analysis (C )and microglia activation was evaluated by histochemical method using rat monoclonal anti-CD11b antibody (D ),and the data were quanti ﬁed respectively (E ,CX3CR1;F ,anti-CD11b).Scale bar,50μm in C and D .Data were represented as mean ±s.e.m.*P o 0.05versus sham;#P o 0.05versus BCAS.IOD,integrated optical density;OD,optical density.CX3CL1/CX3CR1and microglia activation Y Liu et al3corpus callosum(bregma0.26mm)to the anterior aspect of the hippocampus(bregma−0.58mm).Everyﬁfth section(180μm)was cut at10μm and processed for Klüver-Barrera staining.The severity of the lesions in the corpus callosum was graded as normal(grade0), disarrangement of the nerveﬁbers(grade1),formation of marked vacuoles(grade2),or disappearance of myelinatedﬁbers(grade3).26 Water Maze TestThe cognitive function was assessed using a Morris water maze paradigm at day14after BCAS.31The testing was conducted inﬁve consecutive days. Each mouse was subjected to eight trials per day with a5-minute break between trials.A white and circular pool(1.5-m diameter and45-cm deep) wasﬁlled with water to a25-cm depth.Water temperature was maintained at~22°C.A clear Plexiglas platform(11×12cm)was positioned1cm below the surface of water.Mice were placed in the tank facing the wall, randomizing to one of four starting locations(north,south,east,or west), and given90seconds toﬁnd the platform,mount the platform,and remain on it for5seconds.The mice were then placed under a heat lamp to dry before their next trial.The time until the mouse mounted the platform (escape latency)was measured and recorded.The data of Morris water maze test were analyzed by the repeated measures analysis of variance. Statistical AnalysisStatistical analyses were performed using SPSS software for Windows (version10.0;SPSS,Chicago,IL,USA).Data were presented as mean±s.e.m. The difference between or among the means was determined by Kruskal–Wallis test followed by Mann–Whitney test for multiple comparisons or one-way or two-way analysis of variance in appropriate experiments followed by Newman–Keuls post hoc test.A probability value of P o0.05 was considered to be statistically signiﬁcant.RESULTSCX3CR1siRNA Signiﬁcantly Inhibits the Increased Expression of CX3CR1as well as p38MAPK and PKC Induced by Bilateral Common Carotid Artery Stenosis in Mice Brain In VivoTo determine the role of CX3CL1/CX3CR1-mediated activation ofmicroglia and the relevant mechanisms under ischemia,we initially investigated the effects of BCAS on the expression ofCX3CR1and also p38MAPK and PKC in mice brain.Western blot analysis showed that the contents of CX3CR1as well as p38MAPKand PKC in the brain of mice treated with BCAS all were signiﬁcantly higher than those of mice in sham-operated group(Figures1A and1B).Immunostain analysis also showed that BCAS could signiﬁcantly increase CX3CR1expression(Figure1C).Theseevidenced that ischemia induced by BCAS could induce a signiﬁcant increase in the expression of the receptor and molecules involved in the signal transduction pathway.We thenobserved the effects of CX3CR1siRNA on the expression of CX3CR1,p38MAPK,and PKC in the brain of the ischemic mice to ﬁnd out whether the increased expression of p38MAPK and PKC is associated with the expression of CX3CR1.We showed that thelevels of CX3CR1,p38MAPK,and PKC in the mice treated with CX3CR1siRNA and then BCAS were markedly lower than thecorresponding values in the mice treated with BCAS only(Figures 1A and1C).Theﬁnding indicated that the reduced expression of CX3CR1induced by CX3CR1siRNA could lead to a remarkable reduction in the expression of p38MAPK and PKC in the brain of BCAS mice and evidenced that the signiﬁcant increase in the expression of p38MAPK and PKC induced by BCAS is CX3CR1 dependent in mice brain.CX3CR1siRNA Signiﬁcantly Inhibits Microglia Activation Induced by Bilateral Common Carotid Artery Stenosis in Mice Brain In Vivo Second,we investigated whether the ischemia induced by BCAS could activate microglia by immunohistochemical analysis using CD11b,which was used to identify ischemia-induced activation of microglia.32Immunohistochemical data(Figure1D)revealed that microglia have swollen and larger cell bodies with thicker and shorter processes that form thick bundles around an enlarged cell body or a large cell body with almost absent processes in the brain of mice treated with BCAS,indicating that BCAS could lead to activation of microglia.We then evaluated the effects of CX3CR1siRNA(inhibiting CX3CR1expression)on microglia activation induced by BCAS to understand whether the activation is related to the expression of CX3CR1.We showed that the morphologic changes found in microglia in the mice treated with BCAS disappeared mostly in the brain of the mice treated with CX3CR1siRNA and then BCAS.Theﬁndings evidenced that treatment with CX3CR1siRNA could signiﬁcantly attenuate the BCAS-induced microglia activation and also implied that CX3CR1 played a key and essential role in BCAS-induced microglia activation in mice brain.CX3CR1siRNA Signiﬁcantly Inhibits the Increased Expression of Tumor Necrosis Factor-α,Interleukin-1β,and Interleukin-6in Mice Brain In VivoThird,we examined the effects of ischemia induced by BCAS on the expression of microglia cytokines,including TNF-α,IL-1β,and IL-6,in mice brain.Western blot analysis showed that the contents of TNF-αas well as IL-1βand IL-6proteins in the brain of mice treated with BCAS were signiﬁcantly higher than those of mice in the sham-operated group(Figures2A and2B).This showed that ischemia could induce a remarkable increase in the expression of these microglia cytokines.We then explored the effects ofCX3CR1Figure2.CX3CR1siRNA signiﬁcantly reduces the increased expres-sion of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,and IL-6in mice brain in vivo.The mice in bilateral common carotid artery stenosis(BCAS,n=9)or BCAS+siRNA(n=11)group were pretreated with saline or CX3CR1siRNA by intracerebroventricular injection, and then treated with BCAS.The animals in the sham-operated group(n=7)underwent the same surgical procedure as the above two groups without BCAS.At day14after BCAS,the expression of TNF-α,IL-1β,and IL-6was determined by Western blot assay.(A)A representative experiment of Western blot.(B)Quantiﬁcation of expression of proteins.Data were represented as mean±s.e.m. *P o0.05versus sham;#P o0.05versus BCAS.OD,optical density.CX3CL1/CX3CR1and microglia activationY Liu et al 4siRNA on the expression of TNF-α,IL-1β,and IL-6in the brain of the ischemic mice toﬁnd out whether the increased expression of TNF-α,IL-1β,and IL-6is associated with the expression of CX3CR1. We showed that the levels of TNF-α,IL-1β,and IL-6in the mice treated with CX3CR1siRNA and then BCAS were signiﬁcantly lower than those in the mice treated with BCAS only.Theﬁnding suggested that the reduced expression of CX3CR1induced by CX3CR1siRNA could lead to a remarkable reduction in the expression of TNF-α,IL-1β,and IL-6in the brain of BCAS mice and evidenced that the signiﬁcant increase in the expression oftheseFigure3.CX3CR1siRNA signiﬁcantly attenuates white matter lesions and cognitive deﬁcits induced by bilateral common carotid artery stenosis(BCAS)in mice.The mice in BCAS(n=9)or BCAS+siRNA(n=11)group were pretreated with saline or CX3CR1siRNA by intracerebroventricular injection,and then underwent BCAS.The animals in the sham-operated group(n=7)underwent the same surgical procedure as the above two groups without BCAS.At day14after BCAS,grading scores=white matter rarefaction grading scores(A)were evaluated by histochemical method,and cognitive function,Mean Escape latency(seconds)=mean escape latency(seconds;B)assessed by water maze test.(C)A representative histologic analysis of white matter lesion.Data were represented as mean±s.e.m.*P o0.05versus sham;#P o0.05versusBCAS.Figure4.Treatment with oxygen–glucose deprivation(OGD)and CX3CL1signiﬁcantly increase the expression of tumor necrosis factor-α(TNF-α)and interleukin(IL)-1βin BV2microglia in vitro.BV2microglial cells were treated with OGD for4hours in the presence of CX3CL1(0,12.5,25,and50ng/mL),and then the measurements of TNF-αand IL-1βin the medium were conducted using enzyme-linked immunosorbent assay(ELISA)as described in the Materials and Methods section.(A and B)The levels of TNF-αand IL-1βin the medium of BV2microglia treated with OGD for4hours.(C and D)The contents of TNF-αand IL-1βin the medium of BV2microglia treated with OGD plus different concentrations of CX3CL1.Data were represented as mean±s.e.m.(n=5).*P o0.05versus the control(A and B)or0ng/mL CX3CL1(C and D).CX3CL1/CX3CR1and microglia activationY Liu et al5microglia cytokines induced by BCAS is also CX3CR1dependent in mice brain.CX3CR1siRNA Signiﬁcantly Attenuates White Matter Lesions and Cognitive Deﬁcits Induced by Bilateral Common Carotid Artery Stenosis in MiceFinally,we examined the effects of BCAS and CX3CR1siRNA on white matter lesion and cognitive function by histochemical method and water maze test.Histochemical analysis(Figures3A and3C)showed that the white matter rarefaction grading scores in mice of the BCAS group were signiﬁcantly higher than those in mice of the sham-operated group.However,the scores in mice in the BCAS group were signiﬁcantly lower than those in mice in the BCAS+siRNA group.Theﬁndings implied that BCAS could lead to white matter lesion,while CX3CR1siRNA could dramatically suppress the lesion in mice.The results obtained from water maze test(Figure3B)showed that the mean escape latency in mice of the BCAS group were signiﬁcantly longer than those in mice of the sham-operated group.However,the mean escape latency in mice in the BCAS group were signiﬁcantly shorter than those in mice in the BCAS+siRNA group.The data indicated that BCAS could lead to cognitive deﬁcits,while CX3CR1siRNA could signiﬁcantly attenuate the deﬁcits in mice.Theﬁndings also showed that CX3CR1-associated microglia activation plays a detrimental role in ischemic mice under our experimental conditions.CX3CL1Induced a Further Enhancement in Tumor Necrosis Factor-αand Interleukin-1βExpression,Which Could be Signiﬁcantly Attenuated by CX3CR1siRNA or the p38MAPK Inhibitor in Oxygen–Glucose Deprivation–Treated BV2Microglial Cells In Vitro To further investigate the relevant mechanisms involved in the detrimental role of CX3CR1-associated activation of microglia under ischemia,we then investigated the effects of exogenous CX3CL1with CX3CR1siRNA and SB2035080on the expression of microglia cytokines TNF-αand IL-1βin OGD-treated BV2micro-glial cells in vitro.CX3CL1was used in this study because its role in the CX3CR1-associated activation of microglia was well demonstrated.11Theﬁndings(Figures4A and4B)showed that the levels of TNF-αand IL-1βin the culture medium were signiﬁcantly higher in OGD-treated BV2microglia than in control cells, indicating that OGD could induce a signiﬁcant increase in TNF-αand IL-1βrelease from BV2microglia.Also,the addition of different concentrations(0,12.5,25,or50ng/mL)of CX3CL1led to a progressive increase in the contents of TNF-αand IL-1βin the BV2microglial culture medium(Figures4C and4D),implying that CX3CL1has a role to increase TNF-αand IL-1βexpression in BV2 microglia treated without OGD.In addition,the treatment with OGD plus50ng/mL of CX3CL1induced a more signiﬁcantincreaseFigure5.CX3CR1shRNA or SB2035080(SB,a p38MAPK inhibitor)signiﬁcantly reduce the increased expression of tumor necrosis factor-α(TNF-α)and interleukin(IL)-1βinduced by oxygen–glucose deprivation(OGD)and CX3CL1in BV2microglia in vitro.BV2microglial cells were infected with CX3CR1siRNA or negative siRNA for72hours(A and B)or pretreated with20μmol/L of SB2035080only for1hour(C and D), then subjected to OGD for4hours in the presence of50ng/mL of CX3CL1.After the treatments,the measurements of TNF-αand IL-1βwere conducted using enzyme-linked immunosorbent assay(ELISA)as described in the Materials and Methods section.(A and C)TNF-α;(B and D) IL-1β.Data were represented as mean±s.e.m.(n=5).*P o0.05versus the control;#P o0.05versus OGD;and@P o0.05versus CX3CL1+OGD.CX3CL1/CX3CR1and microglia activationY Liu et al6in the expression of TNF-αand IL-1βin BV2microglia as compared with the cells treated with OGD only(Figure5).This increase in the expression of TNF-αand IL-1βinduced by OGD and exogenous CX3CL1could be dramatically suppressed by pretreatment of the cells with CX3CR1siRNA(Figures5A and5B)or the p38MAPK inhibitor(20μmol/L of SB;Figures5C and5D).Theseﬁndings showed that the increased expression of TNF-αand IL-1βinduced by the addition of CX3CL1in OGD-treated BV2microglia is CX3CR1and also p38MAPK dependent.Bilateral Common Carotid Artery Stenosis and Oxygen–Glucose Deprivation Induced a Signiﬁcant Increase in CX3CL1Expression In Vivo and In VitroIn vivo experiments showed that there was a basal level of CX3CL1 expression in mice brain cortex(Figure6A—sham),while BCAS could induce a signiﬁcant increase in CX3CL1expression in this brain region(Figure6A).Immunostaining analysis showed thatthere also was a basal level of CX3CL1expression in normalcultured neurons(Figure6B).Meanwhile,OGD/R treatmentinduced a signiﬁcant increase in CX3CL1level in the neurons(Figure6B)and also in the culture medium(Figure6C).These ﬁndings evidenced that CX3CL1was not only expressed in neurons but also released from neurons in the brain under theconditions of ischemia.DISCUSSIONIn the present study,we showed that ischemia induced by BCAScould lead to white matter lesion,as reﬂected by the signiﬁcantlyincreased white matter rarefaction grading scores and inducecognitive deﬁcits as evidenced by the much longer mean escapelatency in ischemic mice.By using CD11b identiﬁcation,weFigure6.Effects of bilateral common carotid artery stenosis(BCAS)and oxygen–glucose deprivation(OGD)on CX3CL1expression in the brain cortex in vivo and primary cultured neurons in vitro.Mice were treated with BCAS as described in the Materials and Methods section and thenCX3CL1expression was examined by immunostaining(A).Primary cultured neurons were treated with OGD for4hours followed by reoxygenation(R)for24hours.The CX3CL1expression in neurons was then detected by immunostaining(B)and CX3CL1contents in the neuronal culture medium measured by enzyme-linked immunosorbent assay(ELISA;C).Data were represented as mean±s.e.m.(n=3).*P o0.05versus the control.CX3CL1/CX3CR1and microglia activationY Liu et al7。

A Balancing Act for Taxol Precursor Pathways in Ecoli

DOI: 10.1126/science.1191652, 70 (2010);330 Science , et al.Parayil Kumaran Ajikumar Escherichia coliin Isoprenoid Pathway Optimization for Taxol Precursor OverproductionThis copy is for your personal, non-commercial use only.clicking here.colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to othershere.following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles): August 4, 2011 (this infomation is current as of The following resources related to this article are available online at/content/330/6000/70.full.html version of this article at:including high-resolution figures, can be found in the online Updated information and services, /content/suppl/2010/09/27/330.6000.70.DC1.htmlcan be found at:Supporting Online Material /content/330/6000/70.full.html#related found at:can be related to this article A list of selected additional articles on the Science Web sites /content/330/6000/70.full.html#ref-list-1, 4 of which can be accessed free:cites 33 articles This article 1 article(s) on the ISI Web of Science cited by This article has been /content/330/6000/70.full.html#related-urls 1 articles hosted by HighWire Press; see:cited by This article has been/cgi/collection/chemistry Chemistrysubject collections:This article appears in the following registered trademark of AAAS.is a Science 2010 by the American Association for the Advancement of Science; all rights reserved. The title Copyright American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the Science o n A u g u s t 4, 2011w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mIsoprenoid Pathway Optimizationfor Taxol Precursor Overproductionin Escherichia coliParayil Kumaran Ajikumar,1,2Wen-Hai Xiao,1Keith E.J.Tyo,1Yong Wang,3Fritz Simeon,1 Effendi Leonard,1Oliver Mucha,1Too Heng Phon,2Blaine Pfeifer,3*Gregory Stephanopoulos1,2* Taxol(paclitaxel)is a potent anticancer drug first isolated from the Taxus brevifolia Pacific yew tree. Currently,cost-efficient production of Taxol and its analogs remains limited.Here,we report a multivariate-modular approach to metabolic-pathway engineering that succeeded in increasing titers of taxadiene—the first committed Taxol intermediate—approximately1gram per liter(~15,000-fold)in an engineered Escherichia coli strain.Our approach partitioned the taxadiene metabolic pathwayinto two modules:a native upstream methylerythritol-phosphate(MEP)pathway forming isopentenyl pyrophosphate and a heterologous downstream terpenoid–forming pathway.Systematic multivariate search identified conditions that optimally balance the two pathway modules so as to maximize the taxadiene production with minimal accumulation of indole,which is an inhibitory compound found here. We also engineered the next step in Taxol biosynthesis,a P450-mediated5a-oxidation of taxadieneto taxadien-5a-ol.More broadly,the modular pathway engineering approach helped to unlock the potential of the MEP pathway for the engineered production of terpenoid natural products.T axol(paclitaxel)and its structural analogs are among the most potent and commer-cially successful anticancer drugs(1).Taxol was first isolated from the bark of the Pacific yew tree(2),and early-stage production methods required sacrificing two to four fully grown trees to secure sufficient dosage for one patient(3). Taxol’s structural complexity limited its chemical synthesis to elaborate routes that required35to 51steps,with a highest yield of0.4%(4–6).Asemisynthetic route was later devised in whichthe biosynthetic intermediate baccatin III,isolatedfrom plant sources,was chemically converted toTaxol(7).Although this approach and subse-quent plant cell culture–based production effortshave decreased the need for harvesting the yewtree,production still depends on plant-based pro-cesses(8),with accompanying limitations onproductivity and scalability.These methods ofproduction also constrain the number of Taxolderivatives that can be synthesized in the searchfor more efficacious drugs(9,10).Recent developments in metabolic engineer-ing and synthetic biology offer new possibilitiesfor the overproduction of complex natural productsby optimizing more technically amenable micro-bial hosts(11,12).The metabolic pathway forTaxol consists of an upstream isoprenoid pathwaythat is native to Escherichia coli and a het-erologous downstream terpenoid pathway(fig.S1).The upstream methylerythritol-phosphate(MEP)or heterologous mevalonic acid(MV A)pathwayscan produce the two common building blocks,isopentenyl pyrophosphate(IPP)and dimethyl-allyl pyrophosphate(DMAPP),from which Taxoland other isoprenoid compounds are formed(12).Recent studies have highlighted the engi-neering of the above upstream pathways to sup-port the biosynthesis of heterologous isoprenoidssuch as lycopene(13,14),artemisinic acid(15,16),and abietadiene(17,18).The downstream taxadienepathway has been reconstructed in E.coli andSaccharomyces cerevisiae together with the over-expression of upstream pathway enzymes,but todate titers have been limited to less than10mg/liter(19,20).The above rational metabolic engineering ap-proaches examined separately either the upstreamor the downstream terpenoid pathway,implicitlyassuming that modifications are additive(a linearbehavior)(13,17,21).Although this approachcan yield moderate increases in flux,it generallyignores nonspecific effects,such as toxicity of in-termediate metabolites,adverse cellular effects ofthe vectors used for expression,and hidden path-ways and metabolites that may compete with themain pathway and inhibit the production of thedesired binatorial approaches canovercome such problems because they offer theopportunity to broadly sample the parameter spaceand bypass these complex nonlinear interactions(21–23).However,combinatorial approaches re-quire high-throughput screens,which are often notavailable for many desirable natural products(24).Considering the lack of a high-throughputscreen for taxadiene(or other Taxol pathwayintermediate),we resorted to a focused combi-1Department of Chemical Engineering,Massachusetts Institute of Technology(MIT),Cambridge,MA02139,USA.2Chemical and Pharmaceutical Engineering Program,Singapore-MIT Alli-ance,117546Singapore.3Department of Chemical and Bio-logical Engineering,Tufts University,4Colby Street,Medford, MA02155,USA.*To whom correspondence should be addressed.E-mail: gregstep@(G.S.);blaine.pfeifer@(B.P.)Upstream moduleFig.1.isoprenoid pathwaythe flux through thewe targeted reported(dxs,idi,ispD,andexpression by anTo channel theversal isoprenoidtoward Taxolsynthetic operon of downstream genes GGPP synthase(G)and taxadienesynthase(T)(37).Both pathways were placed under the control of induciblepromoters in order to control their relative gene expression.In the E.colimetabolic network,the MEP isoprenoid pathway is initiated by the con-densation of the precursors glyceraldehyde-3phosphate(G3P)and pyruvate(PYR)from glycolysis.The Taxol pathway bifurcation starts from the universalisoprenoid precursors IPP and DMAPP to form geranylgeranyl diphosphate,and then the taxadiene.The cyclic olefin taxadiene undergoes multiple roundsof stereospecific oxidations,acylations,and benzoylation to form the lateintermediate Baccatin III and side chain assembly to,ultimately,form Taxol. REPORTS1OCTOBER2010VOL330SCIENCE 70onAugust4,211www.sciencemag.orgDownloadedfromnatorial approach,which we term “multivariate-modular pathway engineering.”In this approach,the overall pathway is partitioned into smaller modules,and the modules ’expression are varied simultaneously —a multivariate search.This ap-proach can identify an optimally balanced path-way while searching a small combinatorial space.Specifically,we partition the taxadiene-forming pathway into two modules separated at IPP,which is the key intermediate in terpenoid bio-synthesis.The first module comprises an eight-gene,upstream,native (MEP)pathway of which the expression of only four genes deemed to be rate-limiting was modulated,and the second mod-ule comprises a two-gene,downstream,heterolo-gous pathway to taxadiene (Fig.1).This modular approach allowed us to efficiently sample the main parameters affecting pathway flux without the need for a high-throughput screen and to unveil the role of the metabolite indole as in-hibitor of isoprenoid pathway activity.Addition-ally,the multivariate search revealed a highly nonlinear taxadiene flux landscape with a global maximum exhibiting a 15,000-fold increase in taxadiene production over the control,yielding 1.02T 0.08g/liter (SD)taxadiene in fed-batch bioreactor fermentations.We have further engineered the P450-based oxidation chemistry in Taxol biosynthesis in E.coli to convert taxadiene to taxadien-5a -ol and provide the basis for the synthesis of sub-sequent metabolites in the pathway by means of similar cytochrome P450(CYP450)oxida-tion chemistry.Our engineered strain improved taxadiene-5a -ol production by 2400-fold over the state of the art with yeast (25).These ad-vances unlock the potential of microbial pro-cesses for the large-scale production of Taxol or its derivatives and thousands of other valuable terpenoids.The multivariate-modular approach in which various promoters and gene copy-numbers are combined to modulate diverse expression levels of upstream and downstream pathways of taxadiene synthesis is schematically described in fig.S2.A total of 16strains were constructed in order to widen the bottleneck of the MEP pathway as well as optimally balance it with the downstream tax-adiene pathway (26).The dependence of tax-adiene accumulation on the upstream pathway for constant values of the downstream pathway is shown in Fig.2A,and the dependence on the downstream pathway for constant upstream path-way strength is shown in Fig.2B (table S1,cal-culation of the upstream and downstream pathway strength from gene copy number and promoter strength).As the upstream pathway expression increases in Fig.2A from very low levels,tax-adiene production also rises initially because of increased supply of precursors to the overall path-way.However,after an intermediate value further upstream pathway increases cannot be accom-modated by the capacity of the downstream path-way.For constant upstream pathway expression (Fig.2B),a maximum in downstream expressionwas similarly observed owing to the rising edge to initial limiting of taxadiene production by low expression levels of the downstream pathway.At high (after peak)levels of downstream pathway expression,we were probably observing the neg-ative effect on cell physiology of the high copy number.These results demonstrate that dramatic changes in taxadiene accumulation can be obtained fromchanges within a narrow window of expression levels for the upstream and downstream path-ways.For example,a strain containing an ad-ditional copy of the upstream pathway on its chromosome under Trc promoter control (strain 8)(Fig.2A)produced 2000-fold more taxadiene than one expressing only the native MEP path-way (strain 1)(Fig.2A).Furthermore,changing the order of the genes in the downstreamsyn-Fig.2.Optimization of taxadiene production through regulating the expression of the upstream and downstream modular pathways.(A )Response in taxadiene accumulation to changes in upstream pathway strengths for constant values of the downstream pathway.(B )Dependence of taxadiene on the down-stream pathway for constant levels of upstream pathway strength.(C )Taxadiene response from strains (17to 24)engineered with high upstream pathway overexpressions (6to 100a.u.)at two different down-stream expressions (31a.u.and 61a.u.).(D )Modulation of a chromosomally integrated upstream pathway by using increasing promoter strength at two different downstream expressions (31a.u.and 61a.u.).(E )Genotypes of the 32strain constructs whose taxadiene phenotype is shown in Fig.2,A to D.E,E.coli K12MG1655D recA D endA ;EDE3,E.coli K12MG1655D recA D endA with DE3T7RNA polymerase gene in the chromosome;MEP,dxs-idi-ispDF operon;GT,GPPS-TS operon;TG,TS-GPPS operon;Ch1,1copy in chromosome;Trc,Trc promoter;T5,T5promoter;T7,T7promoter;p5,pSC101plasmid;p10,p15A plasmid;and p20,pBR322plasmid. SCIENCEVOL 3301OCTOBER 201071REPORTSo n A u g u s t 4, 2011w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mthetic operon from GT (GGPS-TS)to TG (TS-GGPS)resulted in a two-to threefold increase (strains 1to 4as compared with strains 5,8,11,and 14).Altogether,the engineered strains estab-lished that the MEP pathway flux can be substan-tial if an appropriate range of expression levels for the endogenous upstream and synthetic down-stream pathway are searched simultaneously.To provide ample downstream pathway strength while minimizing the plasmid-born metabolic bur-den (27),two new sets of four strains each were engineered (strains 17to 20and 21to 24),in which the downstream pathway was placed un-der the control of a strong promoter (T7)while keeping a relatively low number of five and 10plasmid copies,respectively.The taxadiene maxi-mum was maintained at high downstream strength (strains 21to 24),whereas a monotonic response was obtained at the low downstream pathway strength (strains 17to 20)(Fig.2C).This ob-servation prompted the construction of two addi-tional sets of four strains each that maintained the same level of downstream pathway strength as before but expressed very low levels of the up-stream pathway (strains 25to 28and 29to 32)(Fig.2D).Additionally,the operon of the up-stream pathway of the latter strain set was chro-mosomally integrated (fig S3).Not only was the taxadiene maximum recovered in these strains,albeit at very low upstream pathway levels,but a much greater taxadiene maximum was attained (~300mg/liter).We believe that this significant increase can be attributed to a decrease in the cell ’s metabolic burden.We next quantified the mRNA levels of 1-deoxy-D -xylulose-5-phosphate synthase (dxs)and taxadiene synthase (TS)(representing the up-stream and downstream pathways,respectively)for the high-taxadiene-producing strains (25to 32and 17and 22)that exhibited varying up-stream and downstream pathway strengths (fig.S4,A and B)to verify our predicted expression strengths were consistent with the actual pathway levels.We found that dxs expression level cor-relates well with the upstream pathway strength.Similar correlations were found for the other genes of the upstream pathway:idi ,ispD ,and ispF (fig.S4,C and D).In downstream TS gene expres-sion,an approximately twofold improvement was quantified as the downstream pathway strength increased from 31to 61arbitrary units (a.u.)(fig.S4B).Metabolomic analysis of the previous strains led to the identification of a distinct metabolite by-product that inversely correlated with taxadiene accumulation (figs.S5and S6).The corresponding peak in the gas chromatography –mass spectrom-etry (GC-MS)chromatogram was identified as indole through GC-MS,1H,and 13C nuclear magnetic resonance (NMR)spectroscopy studies (fig.S7).We found that taxadiene synthesis by strain 26is severely inhibited by exogenous in-dole at indole levels higher than ~100mg/liter (fig.S5B).Further increasing the indole concen-tration also inhibited cell growth,with the level ofinhibition being very strain-dependent (fig.S5C).Although the biochemical mechanism of indole interaction with the isoprenoid pathway is pres-ently unclear,the results in fig.S5suggest a possible synergistic effect between indole and terpenoid compounds of the isoprenoid pathway in inhibiting cell growth.Without knowing the specific mechanism,it appears that strain 26has mitigated the indole ’s effect,which we carried forward for further study.In order to explore the taxadiene-producing potential under controlled conditions for the en-gineered strains,fed-batch cultivations of the three highest taxadiene accumulating strains (~60mg/liter from strain 22;~125mg/liter from strain 17;and ~300mg/liter from strain 26)were carried out in 1-liter bioreactors (Fig.3).The fed-batch cultivation studies were carried out as liquid-liquid two-phase fermentation using a 20%(v/v)dodecane overlay.The organic solvent was intro-duced to prevent air stripping of secreted tax-adiene from the fermentation medium,as indicated by preliminary findings (fig.S8).In defined media with controlled glycerol feeding,taxadiene pro-ductivity increased to 174T 5mg/liter (SD),210T 7mg/liter (SD),and 1020T 80mg/liter (SD)for strains 22,17,and 26,respectively (Fig.3A).Additionally,taxadiene production significantly affected the growth phenotype,acetate accumu-lation,and glycerol consumption [Fig.3,B and D,and supporting online material (SOM)text].Clearly,the high productivity and more robustgrowth of strain 26allowed very high taxadiene accumulation.Further improvements should be possible through optimizing conditions in the bio-reactor,balancing nutrients in the growth medi-um and optimizing carbon delivery.Having succeeded in engineering the bio-synthesis of the “cyclase phase ”of Taxol for high taxadiene production,we turned next to engineer-ing the oxidation-chemistry of Taxol biosynthesis.In this phase,hydroxyl groups are incorporated by oxygenation at seven positions on the taxane core structure,mediated by CYP450-dependent monooxygenases (28).The first oxygenation is the hydroxylation of the C5position,followed by seven similar reactions en route to Taxol (fig.S1)(29).Thus,a key step toward engineering Taxol-producing microbes is the development of CYP450-based oxidation chemistry in vivo.The first oxygenation step is catalyzed by a CYP450,taxadiene 5a -hydroxylase,which is an unusual monooxygenase that catalyzes the hydroxylation reaction along with double-bond migration in the diterpene precursor taxadiene (Fig.1).In general,functional expression of plant CYP450in E.coli is challenging (30)because of the inherent limitations of bacterial platforms,such as the absence of electron transfer machin-ery and CYP450-reductases (CPRs)and trans-lational incompatibility of the membrane signal modules of CYP450enzymes because of the lack of an endoplasmic reticulum.Recently,through transmembrane (TM)engineering and the gener-24487296120T a x a d i e n e (m g /L )Time (h)1234024487296120N e t g l y c e r o l a d d e d (g /L )Time (h)A BC DC e l l g r o w t h (OD 600 n m )Time (h)24487296120A c e t i c a c i d (g /L )Time (h)Fig.3.Fed-batch cultivation of engineered strains in a 1-liter bioreactor.Time courses of (A )taxadiene accumulation,(B )cell growth,(C )acetic acid accumulation,and (D )total substrate (glycerol)addition for strains 22,17,and 26during 5days of fed-batch bioreactor cultivation in 1-liter bioreactor vessels under controlled pH and oxygen conditions with minimal media and 0.5%yeast extract.After glycerol depletes to ~0.5to 1g/liter in the fermentor,3g/liter of glycerol was introduced into the bioreactor during the fermentation.Data are mean of two replicate bioreactors.1OCTOBER 2010VOL 330SCIENCE72REPORTSo n A u g u s t 4, 2011w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mation of chimera enzymes of CYP450and CPR,some plant CYP450s have been expressed in E.coli for the biosynthesis of functional mole-cules (15,31).Still,every plant CYP450has distinct TM signal sequences and electron transfer characteristics from its reductase counterpart (32).Our initial studies were focused on optimizing the expression of codon-optimized synthetic tax-adiene 5a -hydroxylase by N-terminal TM engi-neering and generating chimera enzymes through translational fusion with the CPR redox partner from the Taxus species,Taxus CYP450reductase (TCPR)(Fig.4A)(29,31,33).One of the chi-mera enzymes generated,At24T5a OH-tTCPR,was highly efficient in carrying out the first oxi-dation step,resulting in more than 98%taxadiene conversion to taxadien-5a -ol and the byproduct 5(12)-Oxa-3(11)-cyclotaxane (OCT)(fig.S9A).Compared with the other chimeric CYP450s,At24T5a OH-tTCPR yielded twofold higher (21mg/liter)production of taxadien-5a -ol (Fig.4B).Because of the functional plasticity of taxadiene 5a -hydroxylase with its chimeric CYP450’s en-zymes (At8T5a OH-tTCPR,At24T5a OH-tTCPR,and At42T5a OH-tTCPR),the reaction also yields a complex structural rearrangement of taxadiene into the cyclic ether OCT (fig.S9)(34).The by-product accumulated in approximately equal amounts (~24mg/liter from At24T5a OH-tTCPR)to the desired product taxadien-5a -ol.The productivity of strain 26-At24T5a OH-tTCPR was significantly reduced relative to that of taxadiene production by the parent strain 26(~300mg/liter),with a concomitant increase in indole accumulation.No taxadiene accumulation was observed.Apparently,the introduction of an additional medium copy plasmid (10-copy,p10T7)bearing the At24T5a OH-tTCPR construct dis-turbed the carefully engineered balance in the up-stream and downstream pathway of strain 26(fig S10).Small-scale fermentations were carried out in bioreactors so as to quantify the alcohol production by strain 26-At24T5a OH-tTCPR.The time course profile of taxadien-5a -ol accumulation (Fig.4C)indicates alcohol production of up to 58T 3mg/liter (SD)with an equal amount of the OCT by-product produced.The observed alcohol production was approximately 2400-fold higher than previous production in S.cerevisiae (25).The MEP pathway is energetically balanced and thus overall more efficient in converting either glucose or glycerol to isoprenoids (fig.S11).Yet,during the past 10years many attempts at en-gineering the MEP pathway in E.coli in order to increase the supply of the key precursors IPP and DMAPP for carotenoid (21,35),sesquiterpenoid (16),and diterpenoid (17)overproduction met with limited success.This inefficiency was at-tributed to unknown regulatory effects associated specifically with the expression of the MEP path-way in E.coli (16).Here,we provide evidence that such limitations are correlated with the accumu-lation of the metabolite indole,owing to the non-optimal expression of the pathway,which inhibits the isoprenoid pathway activity.Taxadiene over-production (under conditions of indole-formation suppression),establishes the MEP pathway as a very efficient route for biosynthesis of pharma-ceutical and chemical products of the isoprenoid family (fig.S11).One simply needs to carefully balance the modular pathways,as suggested by our multivariate-modular pathway –engineering approach.For successful microbial production of Taxol,demonstration of the chemical decoration of the taxadiene core by means of CYP450-based oxi-dation chemistry is essential (28).Previous ef-forts to reconstitute partial Taxol pathways in yeast found CYP450activity limiting (25),making the At24T5a OH-tTCPR activity levels an im-portant step to debottleneck the late Taxol path-way.Additionally,the strategies used to create At24T5a OH-tTCPR are probably applicable for the remaining monooxygenases that will require expression in E.coli .CYP450monooxygenases constitute about one half of the 19distinct en-zymatic steps in the Taxol biosynthetic pathway.These genes show unusually high sequence sim-ilarity with each other (>70%)but low similarity (<30%)with other plant CYP450s (36),implying that these monooxygenases are amenable to similar engineering.To complete the synthesis of a suitable Taxol precursor,baccatin III,six more hydroxylation reactions and other steps (including some that have not been identified)need to be effectively engineered.Although this is certainly a daunting task,the current study shows potential by provid-ing the basis for the functional expression of two key steps,cyclization and oxygenation,in Taxol biosynthesis.Most importantly,by unlocking the potential of the MEP pathway a new more ef-ficient route to terpenoid biosynthesis is capable of providing potential commercial production of microbially derived terpenoids for use as chem-icals and fuels from renewable resources.References and Notes1.D.G.Kingston,Phytochemistry 68,1844(2007).2.M.C.Wani,H.L.Taylor,M.E.Wall,P.Coggon,A.T.McPhail,J.Am.Chem.Soc.93,2325(1971).3.M.Suffness,M.E.Wall,in Taxol:Science and 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.(A )TM engineering and construction of chimera protein from taxadien-5a -ol hydroxylase (T5a OH)and Taxus cytochrome P450reductase (TCPR).The labels 1and 2represent the full-length proteins of T5a OH and TCPR identified with 42and 74amino acid TM regions,respectively,and 3represents chimera enzymes generated from three different TM en-gineered T5a OH constructs [At8T5a OH,At24T5a OH,and At42T5a OH constructed by fusing an 8-residue synthetic peptide MALLLAVF (A)to 8,24,and 42AA truncated T5a OH]through a translational fusion with 74AA truncated TCPR (tTCPR)by use of linker peptide GSTGS.(B )Functional activity of At8T5a OH-tTCPR,At24T5a OH-tTCPR,and At42T5a OH-tTCPR constructs transformed into taxadiene producing strain 26.Data are mean T SD for three replicates.(C )Time course profile of taxadien-5a -ol accumulation and growth profile of the strain 26-At24T5a OH-tTCPR fermented in a 1-liter bioreactor.Data are mean of two replicate bioreactors.SCIENCEVOL 3301OCTOBER 201073REPORTSo n 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gratefully acknowledge support by the Singapore-MIT Alliance (SMA-2)and NIH,grant 1-R01-GM085323-01A1.B.P.acknowledges the Milheim Foundation Grant for Cancer Research 2006-17.A patent application that is based on the results presented here has been filed by MIT.P.K.A.designed the experiments and performed the engineering and screening of the strains;W-H.X.performed screening of the strains,bioreactorexperiments,and GC-MS analysis;F.S.carried out the quantitative PCR measurements;O.M.performed the extraction and characterization of taxadiene standard;E.L.,Y.W.,and B.P.supported with cloning experiments;P.K.A.,K.E.J.T.,T.H.P.,B.P.and G.S.analyzed the data;P.K.A.,K.E.J.T.,and G.S.wrote the manuscript;G.S.supervised the research;and all of the authors contributed to discussion of the research and edited and commented on the manuscript.Supporting Online Material/cgi/content/full/330/6000/70/DC1Materials and Methods SOM TextFigs.S1to S11Tables S1to S4References29April 2010;accepted 9August 201010.1126/science.1191652Reactivity of the Gold/Water Interface During Selective Oxidation CatalysisBhushan N.Zope,David D.Hibbitts,Matthew Neurock,Robert J.Davis *The selective oxidation of alcohols in aqueous phase over supported metal catalysts is facilitated by high-pH conditions.We have studied the mechanism of ethanol and glycerol oxidation to acids over various supported gold and platinum beling experiments with 18O 2and H 218O demonstrate that oxygen atoms originating from hydroxide ions instead of molecular oxygen are incorporated into the alcohol during the oxidation reaction.Density functional theory calculations suggest that the reaction path involves both solution-mediated and metal-catalyzed elementary steps.Molecular oxygen is proposed to participate in the catalytic cycle not by dissociation to atomic oxygen but by regenerating hydroxide ions formed via the catalytic decomposition of a peroxide intermediate.The selective oxidation of alcohols with mo-lecular oxygen over gold (Au)catalysts in liquid water offers a sustainable,envi-ronmentally benign alternative to traditional pro-cesses that use expensive inorganic oxidants and harmful organic solvents (1,2).These catalytic transformations are important to the rapidly de-veloping industry based on the conversion of bio-renewable feedstocks to higher-valued chemicals (3,4)as well as the current production of petro-chemicals.Although gold is the noblest of metals (5),the water/Au interface provides a reaction en-vironment that enhances its catalytic performance.We provide here direct evidence for the predomi-nant reaction path during alcohol oxidation at high pH that includes the coupling of both solution-mediated and metal-catalyzed elementary steps.Alcohol oxidation catalyzed by Pt-group metals has been studied extensively,although the precisereaction path and extent of O 2contribution are still under debate (4,6–8).The mechanism for the selective oxidation of alcohols in liquid water over the Au catalysts remains largely un-known (6,9),despite a few recent studies with organic solvents (10–12).In general,supported Au nanoparticles are exceptionally good catalysts for the aerobic oxidation of diverse reagents ranging from simple molecules such as CO and H 2(13)to more complex substrates such as hy-drocarbons and alcohols (14).Au catalysts are also substrate-specific,highly selective,stable against metal leaching,and resistant to overoxidation by O 2(6,15,16).The active catalytic species has been suggested to be anionic Au species (17),cat-ionic Au species (18,19),and neutral Au metal particles (20).Moreover,the size and structure of Au nanoparticles (21,22)as well as the interface of these particles with the support (23)have also been claimed to be important for catalytic ac-tivity.For the well-studied CO oxidation reaction,the presence of water vapor increases the observed rate of the reaction (24–26).Large metallic Au particles and Au metal powder,which are usually considered to be catalytically inert,have consider-able oxidation activity under aqueous conditions at high pH (27,28).We provide insights into the active intermediates and the mechanism for al-cohol oxidation in aqueous media derived from experimental kinetic studies on the oxidation of glycerol and ethanol with isotopically labeled O 2and H 2O over supported Au and Pt catalysts,as well as ab initio density functional theory calcu-lations on ethanol oxidation over metal surfaces.Previous studies indicate that alcohol oxida-tion over supported metal catalysts (Au,Pt,and Pd)proceeds by dehydrogenation to an aldehyde or ketone intermediate,followed by oxidation to the acid product (Eq.1)RCH 2OH À!O 2,catalyst RCH ¼O À!O 2,catalystRCOOH(1)Hydroxide ions play an important role during oxidation;the product distribution depends on pH,and little or no activity is seen over Au cat-alysts without added base.We studied Au par-ticles of various sizes (average diameter ranging from 3.5to 10nm)on different supports (TiO 2and C)as catalysts for alcohol oxidation and com-pared them to Pt and Pd particles supported on C.The oxidation of glycerol (HOCH 2CHOHCH 2OH)to glyceric (HOCH 2CHOHCOOH)and glycolic (HOCH 2COOH)acids occurred at a turnover frequency (TOF)of 6.1and 4.9s −1on Au/C and Au/TiO 2,respectively,at high pH (>13)whereas the TOF on supported Pt and Pd (1.6and 2.2s −1,respectively)was slightly lower at otherwise iden-tical conditions (Table 1).For these Au catalysts,particle size and support composition had negligi-ble effect on the rate or selectivity.In the absence of base,the glycerol oxidation rate was much lower over the Pt and Pd catalysts and no conver-sion was observed over the Au catalysts (Table 1).Moreover,the products detected over Pt and Pd in the absence of base are primarily the intermediate aldehyde and ketone,rather than acids.Department of Chemical Engineering,University of Virginia,102Engineers ’Way,Post Office Box 400741,Charlottesville,VA,22904–4741,USA.*To whom correspondence should be addressed.E-mail:rjd4f@1OCTOBER 2010VOL 330SCIENCE74REPORTSo n A u g u s t 4, 2011w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o m。

gdf-15对急性心肌梗死小鼠心脏血管新生及心功能的影响

基金项目：山西省国际科技合作项目（２０１４０８１０５０－１）作者简介：卫兵艳，女，１９８４－０２生，硕士，实验师，Ｅｍａｉｌ：２８０４６７６５９＠ｑｑ．ｃｏｍ收稿日期：２０１９－０９－２５ＧＤＦ１５对急性心肌梗死小鼠心脏血管新生及心功能的影响卫兵艳１，尚小森２，冯　涛２，樊林花１，轩瑞晶１，刘茂林１，陈小平２，刘田福１＃（１山西医科大学实验动物中心，实验动物和人类疾病动物模型山西省重点实验室，太原　０３０００１；２太原市中心医院心内科；通讯作者，Ｅｍａｉｌ：ｃｘｐ５９０２２３＠１６３；＃共同通讯作者，Ｅｍａｉｌ：１３６０３５１８５７５＠１６３．ｃｏｍ）摘要：　目的　探讨ＧＤＦ１５对急性心肌梗死（ａｃｕｔｅｍｙｏｃａｒｄｉａｌｉｎｆａｒｃｔｉｏｎ，ＡＭＩ）小鼠心脏血管新生及心功能的影响。

　方法　６４只１０周龄的雄性Ｃ５７ＢＬ／６Ｊ小鼠，随机分为４组：假手术组、模型组、ＧＤＦ１５ｓｉＲＮＡ组和ＧＤＦ１５ｃＤＮＡ组，每组１６只。

ＧＤＦ１５ｓｉＲＮＡ组和ＧＤＦ１５ｃＤＮＡ组分别尾静脉注射１００μｌ滴度为１０６－１０７的ＧＤＦ１５ｓｉＲＮＡ病毒和ＧＤＦ１５ｃＤＮＡ病毒，假手术组和模型组给予同等剂量ＰＢＳ。

注射病毒后第３天各组小鼠在麻醉状态下，于左胸４、５肋间隙暴露心脏，模型组、ＧＤＦ１５ｓｉＲＮＡ组和ＧＤＦ１５ｃＤＮＡ组结扎冠状动脉左前降支制作ＡＭＩ模型，假手术组不结扎左前降支，其余操作同模型组。

术后第２１天，小动物超声仪检测各组小鼠心功能，然后每组随机取６只小鼠心脏，ｑＰＣＲ及Ｗｅｓｔｅｒｎｂｌｏｔ测定ＧＤＦ１５表达；另外１０只小鼠心脏，甲醛固定，石蜡包埋后，ＨＥ染色，瘢痕计算法计算梗死面积；ＣＤ３４标记进行免疫组化，经ＩＭＳ图像分析系统处理测定梗死区毛细血管密度；Ｍａｓｓｏｎ染色，ＩＰＰ软件进行分析测定梗死区胶原纤维含量。

　结果　①模型组小鼠左心室舒张末期前壁厚度（ＬＶＡＷｄ）、收缩末期前壁厚度（ＬＶＡＷｓ）、射血分数（ＥＦ）和短轴缩短率（ＦＳ）较假手术组均明显下降，左心室舒张末期内径（ＬＶＥＤｄ）和收缩末期内径（ＬＶＥＤｓ）则显著增加（均Ｐ＜００５）。

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Inhibitory effects of microinjection
2010-07-31
Objective To examine whether microinjectlon of morphine into the rat thaiamle nucleus submedlus (Sin) could depress the bee venom (BV)-induced nociceptive behaviours. Methods In inflammatory pain model induced by BV subcutaneous injection into rat unilateral hind paw, the inhibitory effects of morphine microinjection into thalamic nucleus suhmedius (Sin) on the spontaneous nociecptlve behavior, heat hyperalgesia and tactile ailodynia, and the influence of naioxone on the morphine effects were observed in the rat. Results A single dose of morphine (5.0
Î¼g, 0. 5Î¼L) applied into the Sm ipsilaterni to the BV injected paw significantly depressed the spontaneous paw flinching response. Morphine also significantly increased the heat paw withdrawal iateneies in the bilateral hind paw and the tactile paw withdrawal threshold in the ipsilnteral hind paw 2 hours after BV injection. All these depressive effects could be effectively antagonized by pre-treatment with the opiuld receptor antagonist naloxone (1.0Î¼g, 0. 5Î¼L) in the Sm 5rain prior to morphine administration. Naloxone alone injected to the Sm had no effect on the BV-induecd nociceptive behavior. Conclusion These results suggest that Sm is involved in opioid receptor-mediated antt-nociception in the rat with the BV-induced inflammatory pain. Together with results from previous studies, it is likely that this effect is produced by activation of the Sm-ventrolateral orbital cortex-periaqueductal gray pathway, leading to activation of the brainstem descending inhibitory system and depression of the nodceptive inputs at the spinal cord level.
ä½œ è€…ï¼šJie Feng Ning Jia Jun-yang Wang Xin-ai Song Xiao-ying Li Jing-shi Tang ä½œè€…å •ä½ ï¼šJie Feng,Ning Jia,Xin-ai Song,Xiao-ying Li,Jing-shi Tang(Department of Physiology and Pathoph ysiology,Key Laboratory of Environment and Genes Relatedto Diseases,Ministry of Education,Medical School of Xi'an Jiaotong University,Xi'an 710061)
Jun-yang Wang(Department of Immunology and Pathogenic Biology,Medical School of Xi'an Jiaotong University,Xi'an 710061,China)
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EDITION) å¹´ï¼Œå ·(æœŸ)ï¼š2009 21(2) åˆ†ç±»å ·ï¼šQ426å…³é”®è¯ ï¼šnucl eus submedius morphine hyperaigesia ailodynia bee venom model rat。