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How I treat EBV lymphoproliferation

How I treat

Helen E.Heslop1

1Center for Cell and Gene Therapy,Baylor College of Medicine,The Methodist Hospital and Texas Children’s Hospital,Houston

Epstein-Barr virus(EBV)–associated B-cell lymphoproliferation is a life-threatening complication after hematopoietic stem cell or solid organ transplantation resulting from outgrowth of EBV-infected B cells that would normally be controlled by EBV-cytotoxic T cells.During the past decade,early detection strategies,such as serial

measurement of EBV-DNA load in periph-

eral blood samples,have helped to iden-

tify high-risk patients and to diagnose

early lymphoproliferation.Treatment op-

tions include manipulation of the balance

between outgrowing EBV-infected B cells

and the EBV cytotoxic T lymphocyte re-

sponse and targeting the B cells with

monoclonal antibodies or chemotherapy.

Major challenges remain for de?ning indi-

cations for preemptive therapies and inte-

grating novel and conventional therapies.

(Blood.2009;114:4002-4008)

Introduction

Epstein-Barr virus(EBV)lymphoproliferative disease(LPD)is the result of the outgrowth of EBV-infected B cells that would normally be controlled by an effective EBV-speci?c cytotoxic T-cell response.LPD may occur during both primary and second-ary immune de?ciencies and even in some persons without documented immunode?ciency.In this article,I focus on EBV type III latency B-cell lymphoproliferation,which occurs during the immunosuppression that follows hematopoietic stem cell transplan-tation(HSCT)or solid organ transplantation(SOT).To understand the etiology of these lymphoproliferations and how manipulation of the immune system may be a treatment option,it is important to ?rst understand the biology of EBV.

Biology of EBV

EBV is a latent?-herpesvirus that infects more than90%of the world’s population.Primary lytic infection occurs in the orophar-ynx and may be asymptomatic or present as infectious mononucleo-sis.1EBV is highly immunogenic;and during primary infection, normal persons mount a vigorous humoral and cellular immune response with the cellular component consisting of CD4?and CD8?T cells,which control both primary infection and the periodic reactivations that occur in all EBV-seropositive persons.2 Indeed,analyses using multimers to enumerate EBV-speci?c T cells have shown that up to1%to5%of circulating T cells in a normal EBV-seropositive person may be speci?c for EBV.3,4After clearance of primary infection,EBV persists as an episome in infected B cells,establishing latent infection characterized by the expression of only a limited array of subdominant EBV antigens. There are4types of latency,distinguished by the pattern of EBV antigen expression in infected memory B cells(Figure1).

Most infected circulating memory B cells express no viral antigens(type0latency),allowing them to remain invisible to the host immune system.5EBNA1,which acts on a latent origin of replication,is responsible for coordinating replication of the latent episome in concert with replication of the host cells and is therefore expressed in all types of latency associated with cell division.6Type 1latency is associated with expression of only EBNA-1and is seen in circulating B cells when they proliferate and in Burkitt lym-phoma.In type2latency,LMP1and LMP2are also expressed in addition to EBNA1,and this pattern of expression is seen in germinal center B cells in healthy tonsils.The most immunogenic form of latency is type3,in which all nuclear proteins(EBNAs-1, -2,-3A,-3B,-3C,and-LP)and2membrane proteins(LMP1and LMP2)are expressed together with2small RNAs(EBERs).Type3 latency occurs in B cells immortalized in vitro by EBV into permanently growing lymphoblastoid cell lines.Type3latency B cells are rarely detected in healthy seropositive persons,but their occurrence can be inferred from the high frequency of T cells speci?c for the EBNA3proteins that persist long-term,and it is probable that periodic reactivation to type III latency is rapidly controlled by the EBV-speci?c T-cell response.Patients who are severely immunocompromised,however,so that that EBV-speci?c T cells are severely diminished,may develop B-cell proliferations that express the type3latency pattern of antigens.After SOT or HSCT,these disorders have been labeled posttransplantation lymphoproliferative disease(PTLD).7

Biology of PTLD

Because the immune de?ciency of patients after HSCT or SOT may disrupt the normal balance between latently infected B-cell prolif-eration and the EBV-speci?c T-cell response,the increased number of latently infected B cells8may develop into PTLD,which typically presents with lymphadenopathy or discrete nodules but may be localized to one speci?c site or involve the allograft after SOT.PTLD may also present as more diffuse disease that is more dif?cult to diagnose and may be misinterpreted as a fulminant sepsis syndrome.9

Histologically,PTLD includes a heterogeneous group of lym-phoproliferative disorders ranging from reactive,polyclonal hyper-plasia to aggressive non-Hodgkin lymphomas.A revised classi?ca-tion was published in2008by the World Health Organization and recommends classifying PTLD into4categories:(1)early lesions,

Submitted July1,2009;acceptedAugust5,2009.Prepublished online as Blood First

Edition paper,September1,2009;DOI10.1182/blood-2009-07-143545.

?2009by The American Society of Hematology

4002BLOOD,5NOVEMBER2009?VOLUME114,NUMBER19

(2)polymorphic PTLD,(3)monomorphic PTLD,and (4)classic Hodgkin lymphoma-type PTLD 10(Table 1).All types are associ-ated with EBV .Despite this and earlier efforts to standardize the pathologic classi?cation of PTLD,neither histology nor clonality consistently predicts outcome.11

PTLD after HSCT is predominantly derived from donor B cells and typically occurs within the ?rst 6months after transplantation,before reconstitution of the EBV-speci?c cytotoxic T lymphocyte (CTL)response.It can,however,occur later in the most severely immunocompromised patients.Risk factors include the degree of mismatch between donor and recipient,manipulation of the graft to deplete T cells,and the degree and duration of immunosuppression used to prevent and treat graft-versus-host disease (GVHD).9Although the proliferating B cells are almost always of donor origin,recent reports have described a high incidence of PTLD in pediatric patients who received reduced intensity conditioning regimens that included ATG or Campath,and may be a conse-quence of persisting recipient-derived B cells.12,13

In recipients of SOTs,the severe impairment of T-cell function as result of the immunosuppression required to prevent allograft rejection also places these patients at risk for the development of PTLD.In SOT recipients,PTLD is predominantly of recipient origin.Risk factors for developing this complication include the degree of immunosuppression and development of primary infec-tion after transplantation,so a higher incidence is seen in lung and small bowel transplant recipients as well as in EBV-seronegative pediatric patients receiving a transplant from an EBV-seropositive donor.14Most PTLD occurs in the ?rst year after SOT,and cases occurring later may be EBV-negative and have cytogenetic abnormalities.

Diagnosis

As PTLD may evolve progressively from a polyclonal disorder to a more aggressive monoclonal variant,early diagnosis is important so that treatment can be promptly instituted.There has therefore been much interest in developing predictive assays to identify patients with early disease.Measurement of EBV load by quantita-tive polymerase chain reaction ampli?cation assays can be a sensitive aid to diagnosis,but it is unfortunately not always speci?c for disease onset.Furthermore,different assays use whole blood,serum,or peripheral blood mononuclear cells (PBMCs)and require differing interpretation.When PBMCs are assayed,an elevated EBV DNA may re?ect both EBV in normal B cells (a population that may be expanded in immunosuppressed patients)and EBV in transformed cells.Assays of EBV in serum re?ect virus shedding,which occurs intermittently in normal seropositive persons from epithelium and also from lytically transformed B cells as well as virus released from necrotic transformed cells.Assays measuring whole blood will measure EBV from all these sources.In general,assays using PBMCs are the most sensitive;but in all assays,elevated loads may not always re?ect PTLD.

Initial studies in recipients of HSCT that were selectively T cell–depleted to prevent GVHD suggested that an elevated EBV-DNA load was highly predictive of EBV-PTLD.15,16Follow-up studies,however,which included a broader range of HSCT recipients,showed that only 50%of patients with elevated EBV-DNA subsequently developed PTLD.17Nevertheless,recent evidence-based guidelines from the European Conference in Infec-tions in Leukemia recommend weekly screening of EBV-DNA

for

Figure 1.EBV latent life cycle.Virus enters though mucosal routes (shown is the buccal cavity),then infects normal naive B cells circulating through mucosal sites.Virus expresses type 3latency,which drives B-cell proliferation and expands the infected memory pool.B-cell differentiation into the memory compartments oc-curs in germinal centers driven by type 2latency proteins.Infected memory B cells exiting the germinal center down-regulate viral proteins and are invisible to the immune response.EBNA1is expressed during homeo-static proliferation to maintain the latent viral episome.Virus replication is induced at mucosal sites,and virus is released into the saliva.PTLD indicates posttransplanta-tion lymphoproliferative disease;HD,Hodgkin disease;NPC,nasopharyngeal cancer,and BL,Burkitt lymphoma.

Data from Swerdlow et al.10

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at least3months in high-risk allogeneic HSCT recipients.18 Similarly,whereas up to50%of SOT recipients may have an elevated EBV-DNA load after transplantation,only a much smaller subset will develop PTLD.19In both sets of patients,serial monitoring is therefore important to distinguish patients with a stable-elevated EBV-DNA load from those with increasing EBV-DNA,which may indicate developing https://www.wendangku.net/doc/3a3923984.html,bined monitor-ing of EBV-DNA and EBV-speci?c CTL responses appears to better predict individual patients at risk for PTLD development.20-23 CTL response can be assessed by major histocompatibility com-plex class I peptide-multimer complexes,which enumerate EBV-speci?c CTL,or functionally by measuring interferon-?secretion in response to antigenic stimulation.Unfortunately,neither assay is available outside the research setting.Changes in EBV-DNA load therefore need to be considered along with clinical symptoms,such as fever and lymphadenopathy,and imaging studies,before decid-ing on the need for intervention.The context and risk pro?le are also important in deciding when to intervene,and I would have a lower threshold for intervention in a patient early after HSCT for immune de?ciency than a stable patient several months after kidney transplantation.Similarly,early intervention or even prophy-lactic administration may be warranted where there is a very high risk of developing PTLD,such as a patient with severe GVHD who has received potent T cell–depleting or–suppressing antibodies.24 Treatment options

Treatment options for EBV-PTLD include manipulating the bal-ance between outgrowing EBV-infected B cells and the EBV CTL response,or targeting the B cells with monoclonal antibodies or chemotherapy.

Restoring immune response to EBV

EBV-associated tumors express viral derived antigens and are excellent antigen-presenting cells,expressing high levels of im-mune system costimulatory molecules.Therefore,one therapeutic option is to manipulate the immune system to target and eradicate these malignancies.In HSCT recipients in particular,treatment strategies aim to tilt the balance toward EBV immune responses either by depleting the B-cell population(including EBV-infected B cells)or by augmenting the cellular immune response to EBV.

Reducing immunosuppression.Reducing immunosuppres-sion to restore immune responses to EBV is usually not a useful approach for treating LPD early after HSCT because the patients are profoundly immunosuppressed and the regenerating immune system usually cannot recover fast enough to eradicate the malig-nant cells.When patients are later after transplantation with partial immune recovery,reducing immunosuppression alone may be successful,25and it has also been used as a prophylactic strategy in recipients with increasing EBV DNA levels but no evidence of lymphoma.26In SOTs,where it is a previously normal immune system that has been immunosuppressed,reduction of antirejection drugs may be more useful,and reductions in immunosuppression in response to a rising EBV viral load have indeed reduced the incidence of PTLD in pediatric liver transplant recipients.19Studies of reducing immunosuppression in SOT recipients with overt PTLD,however,have yielded variable results,probably because the process has been empiric with multiple regimens and endpoints used.Because reduction of immunosuppressive therapy carries the risk of graft rejection,graft function must be monitored closely, especially in heart,lung,and liver allograft recipients,for whom no effective alternatives are available if rejection occurs.Neverthe-less,single-center studies report response rates of up to75%in patients treated with this modality alone or in combination with surgery.27,28Factors predicting the failure of reduction of immuno-suppression as a single treatment modality include elevated lactate dehydrogenase,organ dysfunction,and multiorgan involvement.27 The Southwest Oncology Group and the Eastern Cooperative Oncology Group have reported a prospective clinical trial to evaluate reduction of immunosuppression,which mandated a50% reduction in immunosuppressive agents for2weeks followed by a further50%reduction for1week if not in complete remission.In this study,only1of16patients attained a partial response,a much lower response rate than reported in single-center studies.29This strategy is still reasonable in SOT recipients with limited disease, but the patient should be monitored closely so he or she can be transitioned to alternate therapies if needed.

Unmanipulated donor T cells.Because most HSCT cell donors are EBV seropositive,an EBV-speci?c T-cell response can often be provided simply by infusing unmanipulated donor lympho-cytes.Although this approach has shown clinical ef?cacy in HSCT patients with established PTLD with response rates of more than 70%,it carries a signi?cant risk of inducing severe or fatal GVHD, as the frequency of alloreactive T cells in the cell product is more than a log higher than the frequency of virus-reactive T cells.30,31 One investigational approach to circumvent this problem is to transduce T cells with a suicide gene,such as the thymidine kinase gene,which can be activated by infusion of ganciclovir should the recipient develop GVHD.This approach has proved effective in early phase studies and is being evaluated in a phase3licensing trial in Europe.32

EBV-speci?c T cells.The problem of alloreactivity can also be overcome by infusing EBV-speci?c CTLs generated using EBV-transformed lymphoblastoid B-cell lines,which will effectively present the viral antigens expressed on the tumor cells as antigen-presenting cells.We have given donor-derived EBV-speci?c CTL to more than100patients after HSCT and found them to be highly effective as prophylaxis in high-risk patients with a history of PTLD or patients receiving selective T-cell depletion.33,34They have also proved effective as treatment for overt PTLD in more than80%of patients,35a?nding con?rmed by other investigators, who have also shown that EBV-CTLs are active in transplant recipients with rituximab-resistant PTLD.36-39In the SOT recipient, there are additional challenges because patients remain on long-term immunosuppression.Several studies have nonetheless shown that it is possible to generate autologous EBV CTLs from these patients,which will restore short-term EBV-speci?c immunity that controls disease progression.Long-term persistence,however,has not been apparent in patients who continue to receive immunosup-pression.40-42In addition,both these approaches are currently con?ned to experimental protocols,and additional drawbacks are the time(2-3months)and facilities required for CTL production.

One solution to make this strategy more accessible to a larger number of patients is to bypass the development of separate lines for each affected patient by developing a bank of partially human leukocyte antigen(HLA)–matched,allogeneic lines,which can be readily and rapidly available.Investigators in Great Britain re-ported a phase2clinical trial using partially HLA-matched

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allogeneic CTL for PTLD therapy in a cohort of HSCT and SOT recipients who had failed to respond to conventional PTLD therapy,obtaining a64%(21of33)and52%(17of33)response rate at5weeks and6months,respectively.43Patients with closer HLA-matching donors showed better responses at6months. Targeting B cells

Antibody therapy.As most cases of PTLD arise in donor-or recipient-derived B cells,one strategy for prevention and treat-ment is to eliminate EBV-infected B cells.Antibody therapy targets B cell–speci?c surface antigens present on the EBV-transformed malignant cells.The most widely used antibody is rituximab,a chimeric murine/human monoclonal anti-CD20 antibody.Rituximab has been used as prophylaxis and treatment for PTLD after HSCT,with initial response rates between55% and100%,44-47a range that probably re?ects differences in the treated patient populations.Rituximab also has activity in PTLD after SOT,in which response rates of44%to100%have been reported in several small studies.48-51The results of these studies are summarized in Table2.Fewer late-phase studies have been reported,but a recent phase2clinical trial using rituximab to treat PTLD reported a response rate of44%at day80.This trial included patients whose only previous therapy was reduction of immunosuppression but excluded patients with central nervous system(CNS)PTLD.52

Because CD20expression is not con?ned to the malignant cells, normal B cells are also destroyed.This can be a signi?cant concern in patients who are already immunosuppressed,and fatal viral infections have been reported after rituximab therapy.53As ritux-imab can deplete B cells for more than6months in these already immunosuppressed patients,it should be used as preemptive therapy for PTLD only where there is a strong probability of subsequent lymphoma.An additional concern is that,when used as therapy,it does not restore the cellular immune response to EBV, which is a crucial requirement if EBV-mediated B-cell proliferation is to be controlled long-term.48This may not be a major problem in most HSCT recipients,in whom recovery of a donor-derived immune response should provide long-term protection,but it is a concern in SOT patients who remain on long-term immunosuppres-sion.Indeed,a long-term follow-up study of SOT patients treated with rituximab showed that57%had progressive disease at 12months,54whereas another study showed PTLD recurrence in 50%.48A?nal concern is that only one antigen is targeted and antigen-loss tumor cell variants may be selected.

Antiviral agents.Antiviral treatment for lytic EBV infection currently makes use of nucleoside analogs,which target the virus-speci?c enzyme,thymidine kinase(TK)expressed in lyti-cally infected cells.The lack of viral TK expression in EBV-positive tumors during viral latency,however,makes antiviral therapy alone ineffective as an antineoplastic therapy.Antiviral agents may have a role in prophylaxis in EBV-seronegative SOT recipients as they can block EBV production in donor B cells and subsequent infection of recipient B cells.55Moreover,long-term prophylaxis with antiviral agents or intravenous immunoglobulin may decrease the incidence of PTLD by limiting intercellular virus transmission;several centers use prophylactic acyclovir or intrave-nous immunoglobulin in the?rst6months after allograft,during which time the immunosuppression is most intense.56Once PTLD is established,however,antiviral agents will have no effect on the growth of type II latency cells that are already transformed.One possible means of exploiting the susceptibility of lytic cycle cells to acyclovir or ganciclovir is to induce reexpression of the viral-associated TK or the lytic cycle of EBV infection,using chemo-therapy57or arginine butyrate.58A trial of arginine butyrate and ganciclovir included6patients with PTLD and encouragingly reported2complete and3partial responses.58

Radiation therapy and surgery.When PTLD is con?ned to one site,radiation and/or surgery can effectively control local disease.Indeed,in one report on the outcome of PTLD occurring late after SOT,all patients treated with surgery or radiotherapy for limited disease and reduction of immunosuppression obtained sustained complete responses.59Surgery and radiation also have a role in managing local complications of PTLD,such as compres-sion of vital organ structures.

Chemotherapy.Chemotherapy with regimens used in lym-phoma therapy,such as CHOP(cyclophosphamide,doxorubicin, vincristine,and prednisone),remains a therapeutic option for patients who fail to respond to immune manipulation or rituximab. One concern is that PTLD patients may be more susceptible to chemotherapy toxicity(particularly HSCT recipients who have already received intensive chemotherapy),and they are also at increased risk of infection because of their preexisting immune suppression.Although high toxicity rates were seen in initial reports,more recent reports have shown better outcomes.A recent retrospective analysis of standard CHOP in26adults with PTLD demonstrated an overall response rate of65%and median overall and progression-free survivals of13.9and42months,respectively, after a median follow-up of8.8years.60PTLD generally remains chemotherapy-sensitive after progression or failure to respond to rituximab used as?rst-line therapy,and CHOP salvage therapy can achieve an overall response rate up to70%in these patients.61By comparison,EBV-unrelated,PTLD after SOT has a lower response rate but may respond to high-dose chemotherapy followed by HSC rescue.62To reduce toxicity from chemotherapy,a study evaluated lower-dose chemotherapy with cyclophosphamide and prednisone in36pediatric patients who had failed frontline therapy and reported an excellent overall response rate of83%.63This lower-dose regimen therefore appears effective as salvage therapy for children with PTLD.

PTLD involving the CNS can present a particular challenge as multiple lesions can be present and the prognosis is poor.64,65In the largest reported series,the best outcomes were seen with radiation for isolated CNS disease.65Responses have also been reported with reduction in immunosuppression and rituximab,although this agent crosses the blood-brain barrier poorly.66Chemotherapy with high-dose methotrexate has also induced responses in a report of

Initial response rates to rituximab in several small trials in patients with PTLD after transplantation.

CR indicates complete response;and PR,partial response.EBV LYMPHOPROLIFERATION4005

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administration to 4children with CNS PTLD after liver transplantation.67

Hydroxyurea is another less toxic option that can eradicate episomal DNA elements,which may be required for the continued growth of EBV-associated lymphomas.There are several case reports showing the activity of this agent in EBV-associated B-cell lymphoproliferations,particularly with CNS involvement,but these results have not yet been con?rmed in prospective trials.68,69Rapamycin also has activity against EBV-transformed B cells in vitro,and there are anecdotal reports of using the agent as a substitute immunosuppressive agent that can treat PTLD.70

Model treatment schema

Perhaps one of the most contentious issues with PTLD is not how but when to treat.My general philosophy is to reserve preemptive

treatment or treatment based on EBV DNA alone for very-high-risk situations,such as a patient with a diagnosis of X-linked lympho-proliferative disease or Wiskott-Aldrich syndrome.71Treatment for rejection or GVHD with an intensive anti-T-cell antibody that does not also target B cells would also place a patient in a high-risk category,in which preemptive treatment of a rising EBV DNA in the absence of other symptoms or signs could be considered.24

In other settings,my goal would be to diagnose PTLD at an early stage so it could be treated promptly using the diagnostic tests reviewed in Table 3.I would therefore monitor EBV DNA in high-risk recipients at 1to 2week intervals.In patients who develop elevated levels in the absence of symptoms,modulation of immunosuppressive therapy (if feasible)is a reasonable strategy.I would have a low threshold for imaging studies in patients with elevated EBV DNA who also had clinical symptoms,such as fever,that were consistent with the diagnosis.If imaging or physical examination shows enlarged lymph nodes or other evidence of disease,it is helpful to biopsy sites of involvement to con?rm the diagnosis and exclude other infections that may have a similar imaging appearance.Biopsy also allows con?rmation of CD20expression,allowing rituximab to be used.In patients with later PTLD after SOT,cytogenetic studies are also helpful in identifying lymphomas that will be less susceptible to immune manipulation and should be treated like lymphomas arising in immunocompetent persons.A monitoring and treatment algorithm is outlined in Figure 2.In patients with localized disease,surgery alone or with reduction of immunosuppression may provide suf?cient control of disease.In SOT patients with early disease,a trial of reduction in immunosuppression alone is reasonable,with the addition of rituximab in patients who fail to respond or progress.SOT patients with higher-grade disease and HSCT recipients unless they have some evidence of recovery of an EBV-speci?c T-cell response should proceed to rituximab.

In patients with more extensive higher-grade disease or patients who progress after initial maneuvers,the options include chemo-therapy or T-cell therapies.Donor T cells are only an option in HSCT recipients,whereas EBV-CTLs are investigational and will be restricted to centers participating in clinical trials.Nevertheless,given the morbidity of standard chemotherapy,particularly in HSCT recipients who have often received extensive previous chemotherapy,I think it is reasonable to consider cell therapy at this stage.

In conclusion,one problem in developing a treatment algorithm for treating EBV-associated PTLD is the lack of later-phase trials from which to develop evidence-based guidelines.Hopefully,as newer therapies,such as allogeneic EBV CTLs,and more targeted chemotherapy regimens are evaluated,more de?nitive trials to treat EBV lymphoproliferations will be designed and completed so that treatment in the future can be more

evidence-based.

Figure 2.Monitoring and treatment algorithm.Professional illustration by Debra T .Dartez.

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Acknowledgments

The author thanks Dr Cliona Rooney for her contribution to Figure1.

This work was supported by the National Institutes of Health (grants PO1CA94237,P50CA126752)and the Leukemia& Lymphoma Society.Authorship

Contribution:H.E.H.wrote the manuscript.

Con?ict-of-interest disclosure:The author declares no compet-ing?nancial interests.

Correspondence:Helen E.Heslop,Center for Cell and Gene Therapy,1102Bates St,Suite1620,Houston,TX77030;e-mail: hheslop@https://www.wendangku.net/doc/3a3923984.html,.

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4008HESLOP BLOOD,5NOVEMBER2009?VOLUME114,NUMBER19

online September 1, 2009

originally published doi:10.1182/blood-2009-07-1435452009 114: 4002-4008

Helen E. Heslop

How I treat EBV lymphoproliferation

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高度组织亲和力的雷米普利

高度组织亲和力的雷米普利治疗高血压、糖尿病的应用展望郭冀珍陶波摘要本文重点介绍雷米普利的作用机制、作用特点及在治疗高血压、糖尿病中的应用。认为雷米普利不仅能有效地治疗轻、中度高血压及充血性心力衰竭，还能改善肝糖代谢、减少糖尿病及高血压对肾脏的损害。该药为亲脂性，对组织亲和力强、副作用少，尤适用于肥胖的高血压病和糖尿病病人。本文重点阐述雷米普利的亲脂性和对组织高度亲和力的作用。关键词雷米普利；血管紧张素转换酶抑制剂；高血压；糖尿病雷米普利(ramipril)为一新型的非巯基血管紧张素转换酶(ACE)抑制剂的前体药物。在体内吸收后水解成雷米普利拉(ramiprilat)而发挥作用。ACE抑制剂除了作用于肾素-血管紧张素系统(renin-angiotensin system,RAS),抑制ACE活性，减少血管紧张素 II(angiotensin II,ATII)的生成外，还能抑制激肽酶II(kininase II,K II)引起的激肽降解，ACE-K II是一种双肽碳氧化酶，虽然ACE抑制剂的降压及靶器官的保护作用主要通过RAS起作用，但是临床和动物试验均发现ACE抑制剂部分依赖于抑制缓激肽降解而起作用；尤其重要的是，组织中的RAS通过作用于激肽释放酶-激肽系统 (kallikrein-kinin system,KKS)产生局部效应［1-4］。雷米普利的作用机制 1 激肽释放酶与激肽的作用激肽释放酶能将激肽原(kininogen)水解为激肽，在血浆和组织中存在2种激肽释放酶，一种是以非活性状态存在于血浆中，即前激肽释放酶，它能被血浆中的凝血因子XII所激活。另一种组织中的激肽释放酶是一群结构相似的蛋白酶。激肽可被体循环中的激肽酶I或局部组织中的激肽酶II迅速降解［1，2］。激肽受体包括B1和B2受体2种，他们分布于不同的组织中，B1受体与血压变化关系不大，而B2受体与血管扩张有关，可能影响血压的变化。因此，组织RAS可能主要通过B2受体产生效应。激肽与受体结合激发内皮介质的释放，如一氧化氮(NO)、前列环素(PGI2)和内皮派生的超极化因子(EDHF)，除了可能参与降压外还能引起血管扩张，如冠状动脉和肾动脉等。缓激肽还通过诱导凋亡，减少平滑肌细胞的数目抑制血管平滑肌的生长和增生［3，4］。 2 缓激肽B2受体拮抗剂与雷米普利的关系自从缓激肽B2受体拮抗剂——HOE140 等应用于科研，不少文献已报道雷米普利对KKS的影响，对降压、靶器官保护及对代谢的作用等方面有了进一步的认识，给予HOE140后，长期应用雷米普利所引起的内皮介导的血管舒张效应明显减弱，表明这种血管舒张效应主要取决于缓激肽的作用。通过对实验动物的研究发现，缓激肽对高肾素型的高血压大鼠的降压效果较好，而对于自发性高血压大鼠的降压效果不佳，临床试验还缺乏这方面的研究。其机制也有待进一步阐明，可能与体内ATII与NO的平衡状态有关［5］。雷米普利能提高内源性激肽作用达50倍，用雷米普利能明显抑制大鼠颈动脉内皮细胞的增殖，而与缓激肽B2受体拮抗剂(HOE140等)合用，则明显减弱这种效应，说明缓激肽有拮抗细胞增殖的作用［5］。另外，单用雷米普利有降低血压(P<0.001)、降低胰岛素水平(P<0.05)与降低三酰甘油(TG)水平 (P<0.001)的作用，但如与HOE140合用则仅有降压作用，提示雷米普利对血脂与血糖代谢的影响高度依赖缓激肽的积累效应，而与ATII的水平关系较小［6，7］。在氧化低密度脂

高亲和力谷氨酸转运体

?综述?高亲和力谷氨酸转运体3 杨　如　杨雄里(中国科学院上海生理研究所,上海200031) 摘要　高亲和力谷氨酸转运体主要位于神经元和胶质细胞的细胞膜上,能逆浓度梯度从胞外向胞内摄取谷氨酸,中止谷氨酸能传递,使胞外谷氨酸浓度保持在较低水平,以保护神经元不受谷氨酸的毒性影响。近年来,随着高亲和力谷氨酸转运体的克隆,有关研究迅速发展。本文从高亲和力谷氨酸转运体的克隆、分子结构特征、表达分布、生理功能、结构2功能关系等方面对近年的进展加以综述。关键词　谷氨酸转运体;兴奋性氨基酸;神经递质学科分类号　Q424 High2Aff inity G lutamate T ransporters　YAN G Ru,YAN G Xiong2Li(S hanghai In2 stit ute of Physiology,Chi nese A cademy of Sciences,Shanghai200031) Abstract　High2affinity glutamate transporters are located predominantly in the plasma membrane of neurons and glial cells.They have the capacity to take up glutamate from the extracellular space into the cells against its concentration gradient to terminate gluta2 matergic transmission and to keep the extracellular glutamate concentration at low levels to protect neurons from glutamate toxicity.As glutamate transporters were recently cloned,the research in this field has been greatly advancing.This article focuses on re2 cent progress in the study of molecular structure,distribution of expression,physiologi2 cal significance,structure2function relationships of these transporters. K ey w ords　G lutamate transporter;Excitatory amino acid;Neurotransmitter 谷氨酸是中枢神经系统兴奋性突触传递的主要神经递质。由于胞外不存在谷氨酸代谢酶,谷氨酸清除的主要途径之一是由高亲和力谷氨酸转运体摄取谷氨酸。高亲和力谷氨酸转运体(以下简称为谷氨酸转运体)分为G LAST(或简称EAA T1)、G L T1(EAA T2)、EAAC1 (EAA T3)、EAA T4和EAA T5等5个类型[1～5],虽然它们在其它组织也有分布,但主要位于神经元和胶质细胞的细胞膜上,其作用是逆浓度梯度从胞外将谷氨酸摄入神经元和胶质细胞内,在突触部位适时中止谷氨酸能传递,并使胞外谷氨酸浓度保持在较低水平,保护神经元不受谷氨酸的毒性影响。这种转运将氨基酸摄取与同向转运Na+和逆向转运K+相偶联,又与同向转运H+(或逆向转运OH-)相偶联。一般认为,转运体只起转运递质的作用,但近来发现,有些谷氨酸转运体有类似通道的特性,对氯离子有很高的通透性。 3　国家重点基础研究规划(G1999054000)、国家自然科学基金(39770256)和上海生命科学研究中心资助课题

高亲和力单克隆抗体制备问题及对策

高亲和力单克隆抗体制备问题及对策一、单抗制备-骨髓瘤细胞SP2/0培养过程中遇到的问题及对策 1，细胞长的慢或细胞死亡正常生长情况下，细胞一天传代一次，生长越好，贴壁越多对策：①培养基配方不对；②接种密度太低，低于10的4次方；③污染了支原体或者其他不明细菌，支原体的现状是出现小黑点；细菌污染是浑浊；霉菌污染是出现白色肉眼可见菌落；不明细菌污染出现砂状平铺背景，视野发暗。④细胞瓶刷洗不干净 2，细胞形态异常正常生长情况下，细胞浑圆，透亮，成对数生长，生长越好，贴壁多，悬浮少对策：①培养基配方不对；②接种密度太低，低于10的4次方；③污染了支原体或者其他不明细菌，支原体的现状是出现小黑点；细菌污染是浑浊；霉菌污染是出现白色肉眼可见菌落；不明细菌污染出现砂状平铺背景，视野发暗。④细胞瓶刷洗不干净二、单抗制备-细胞融合筛选过程中遇到的问题及对策 1，融合率低，阳性孔少 ①免疫的问题。由于免疫原性不强或者免疫途径不当造成免疫弱，效价低。 ②融合过程中温度、时间、PEG分子量，作用时间等。 ③脾细胞是否取出了足够多的细胞，有无组织碎片干扰。 ④融合前骨髓瘤细胞生长状态是否完好，细胞是否浑圆，透亮，成对数生长。

2，单抗亲和力整体低 ①免疫的问题。免疫途径不当或者免疫原问题。 ②融合细胞筛选过程出现遗漏，筛选方法不当或检测方法不当，造成没有筛到高亲和力的融合细胞。 ③由于亚克隆细胞生长环境不合适，导致融合细胞染色体丢失，分泌特性改变，亲和力从高变低，这时就需要及时更换培养液，当细胞增长到一定密度的时候，培养基就开始变颜色，影响融合细胞的生长，造成染色体丢失；控制好细胞的密度，密度过大使新加入的培养基不至于很快被消耗，影响融合细胞的生长，造成染色体丢失；不要过于频繁操作细胞，当细胞生长密度不是很大的时候，不要频繁操作，否则细胞容易出现死亡或者生长形态发生明显变化。操作细致，防止细胞被支原体等微生物污染。 3，抗体亲和力从高变低 ①没有及时更换培养液，当细胞增长到一定密度的时候，培养基就开始变颜色，影响融合细胞的生长，造成染色体丢失或者性状改变。 ②控制好细胞的密度，密度过大使新加入的培养基不至于很快被消耗，影响融合细胞的生长，造成染色体丢失或者性状改变。 ③过于频繁操作细胞，当细胞生长密度不是很大的时候，不要频繁操作，否则细胞容易出现死亡或者生长形态发生明显变化。 ④细胞被支原体等微生物污染。 ⑤亚克隆次数超过5次，导致细胞生长环境经常被改变，弱的阳性杂交瘤细胞染色体丢失或者性状改变。三、单抗腹水制备过程中遇到的问题及对策 1，腹水少或者无腹水产生