The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xu, J.
Right arrow Articles by Menezes, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xu, J.
Right arrow Articles by Menezes, J.
The Journal of Immunology, 2000, 164: 2815-2822.
Copyright © 2000 by The American Association of Immunologists

Analysis and Significance of Anti-Latent Membrane Protein-1 Antibodies in the Sera of Patients with EBV-Associated Diseases

Jingwu Xu*, Ali Ahmad*, Mario D’Addario*, Laurent Knafo*, James F. Jones{dagger}, U. Prasad{ddagger}, R. Dolcetti§, E. Vaccher§ and José Menezes*

* Laboratory of Immunovirology, Pediatric Research Center and Department of Microbiology and Immunology, University of Montreal and Ste.-Justine Hospital, Montreal, Quebec, Canada; {dagger} Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO; {ddagger} Department of Otorhinolaryngology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; and § Divisions of Experimental Oncology, and Medical Oncology and AIDS, Centro di Riferimento Oncologico, Aviano (PN), Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Anti-latent membrane protein-1 (LMP-1) is an EBV-encoded type III integral membrane protein with oncogenic potential that is expressed most consistently in various EBV-associated malignancies. Unlike many other EBV proteins, LMP-1 Abs have rarely been demonstrated in EBV-associated disease conditions. We established a high level LMP-1-expressing cell clone and used it for the detection, quantitation, and characterization of these Abs in various human sera in immunoblots and ELISA. Our results demonstrate that, in contrast to the commonly held notion, LMP-1 induces significant humoral immune responses in EBV-associated malignant conditions especially in nasopharyngeal carcinoma (NPC) patients in whom >70% sera are positive for these Abs, and their titers correlate with the clinical condition of the tumors. Interestingly, anti-LMP-1 Abs of IgA isotype were found only in NPC patients. These Abs were absent from the sera of infectious mononucleosis and chronic EBV infection patients, whereas a small fraction (~5%) of the healthy, EBV-seropositive individuals were positive for them; however, their OD values were much lower than those of NPC patients. These studies demonstrate, for the first time, the potential significance of LMP-1-specific Abs for the diagnosis and prognosis of EBV-associated malignancies, especially of NPC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The EBV is a highly prevalent herpesvirus in humans, in whom it may induce several lymphoproliferative diseases (1, 2, 3). Primary infections of EBV usually occur in early childhood, and these infections are often self-limiting and present with mild symptoms. The infected hosts, however, never completely eliminate the virus, which persists in them mainly in a latent form for their entire lives. In industrialized western countries, where primary infections are delayed until adolescence, EBV is the major cause of infectious mononucleosis (IM),3 a mild lymphoproliferative disorder accompanied by clinical symptoms of fever, malaise, pharyngitis, rashes, etc. (2, 3). A proportion of the infected persons develops chronic EBV infections; they present with a variety of disease symptoms and have unusually high titers of Abs to the lytic and latent cycle viral Ags. The virus replication in these individuals remains high. In the individuals with X-linked lymphoproliferative syndrome, EBV infections are usually fatal (2).

EBV has been causally associated with many human tumors, e.g.: nonkeratinizing nasopharyngeal carcinoma (NPC), which is prevalent in Southeast Asia, North Africa and in certain indigenous populations of North America; endemic Burkitt’s lymphoma in Africa and with the classic Hodgkin’s disease (HD) (2, 3). Recently, EBV has been found associated with T cell lymphomas, NK cell granulomas of the nasal septum, gastric tumors, and B cell lymphomas of AIDS patients (1, 2, 3). In the latter patients, EBV also causes oral hairy leukoplakia characterized by the localized lesions in the oral cavity, especially on tongue, in which intensive viral replication is taking place (2). In vitro, EBV infects and readily immortalizes human B cells into continuously growing lymphoblastoid cell lines (2).

Latent membrane protein-1 (LMP-1) is one of the limited number of EBV Ags that are expressed in the latent EBV infection. This infection is exemplified by EBV-immortalized B cells (1). In the latently infected cells, EBV exists as an episome that replicates once with each cell division and expresses only a few genes, i.e., LMP-1, -2A, -2B, six EBV nuclear Ags, and two small polyadenylated EBV RNAs (EBERs) (1, 3). In addition to its role in latent infection (see below), LMP-1 (hereafter referred to as LMP) also seems to play a role in lytic EBV infection by acting as an antiapoptotic protein and thereby delaying the cell death until all the viral structural and nonstructural proteins are expressed, and virions bud off the infected cells (1, 4). Because LMP has been detected in virions, it may also play a role in the viral infection process (5).

LMP is a 63-kDa type III protein which carries six hydrophobic transmembrane regions and occurs as aggregated patches spanning the surface of EBV-infected and/or-immortalized cells. It resembles TNFR family of proteins (6). Its intracellular carboxy-terminal cytosolic region interacts with signaling molecules that are involved in the pathways activated by the TNFR family members which include CD40 (3, 7, 8, 9, 10, 11). By this virtue, LMP acts as a constitutively activated CD40 receptor in B cells. LMP transforms rodent fibroblasts and is essential for the EBV-induced immortalization of B cells (1, 3, 12). It induces activation of NF-{kappa}B, c-JNK, and AP-1, and the expression of antiapoptotic genes Bcl2, Mcl-1, and A20 in different cell types (4, 13, 14). In LMP-transfected cells, it induces several phenotypic changes characteristic of the EBV-infected cells, e.g., CD23, epidermal growth factor receptor, certain adhesion molecules, etc. (3, 13, 14). Recently, LMP was shown to induce the expression of cyclin D2 and hyperphosphorylation of pRb, rendering cells unresponsive to the growth-inhibitory effects of TGF-ß1 (15). These studies strongly suggest a role of LMP-1 in the EBV-associated lymphoproliferative diseases and malignancies. This is further supported by the consistent expression of this protein in many EBV-associated tumors.

The EBV-infected subjects mount a vigorous antiviral cellular and humoral immune responses (reviewed in Refs. 16, 17). These responses, particularly cellular ones, are thought to keep the viral replication under control in healthy subjects. The detection of humoral responses (i.e., Abs) against various viral Ags has been very useful for the diagnosis and prognosis of EBV-associated disease conditions. LMP-specific cellular immune responses have been well documented (16, 17). However, information on anti-LMP Abs is scanty despite the fact that this protein is expressed on the surface of EBV-infected/immortalized cells and may be targeted by such Abs. The detection of these Abs may be helpful not only in the diagnosis and prognosis of EBV-associated diseases but may also provide insights about their pathogenetic processes. We report here our studies on the detection, analysis, and significance of LMP-specific Abs in different EBV-associated diseases and show for the first time that these Abs can readily be demonstrated in the sera of patients with EBV-associated malignancies and that anti-LMP IgA may be of prognostic value in patients with advanced stages of NPC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Expression of LMP-1 in BJA-B

LMP-1 was expressed in an EBV-genome negative human B cell line BJA-B derived from Burkitt’s lymphoma (18). For this purpose, a pGEM2 (Promega, Madison, WI)-based expression plasmid pIgLMP-1 (provided by Dr. Nancy Raab-Traub, University of North Carolina, Chapel Hill, NC) was transfected into these cells by electroporation as described earlier (19). This plasmid has an Ig promoter/enhancer 5' to two tandemly arranged copies of the 3.0-kb LMP-1 genomic sequences and carries a neomycin resistance marker for selection of transfected eukaryotic cells. The control plasmid (pIgNeo) was obtained from pIgLMP-1 after deleting the LMP sequences. The transfected cells were cloned by limiting dilution in the selection medium, i.e., RPMI 1640 containing 10% FBS and 1 mg per ml of geneticin (Life Technologies, Burlington, ON, Canada). The transfected cell clones were screened for LMP-1 expression by immunofluorescence using S12 (provided by Dr. Elliott Kieff, Harvard Medical School, Boston, MA) and CS1-4 (Dako, Glostrup, Denmark) Abs. The selected clones were maintained in the selection medium, and their lysates were used to detect anti-LMP-1 Abs in the sera (see below).

Serum samples

Serum samples for the detection of anti-LMP Abs were obtained after informed consent from the following groups of individuals: category 1, healthy EBV seronegatives (HN), 3; category 2, healthy EBV seropositives (HI), 20; category 3, IM, 15; category 4, chronic EBV infection (CEI), 25; category 5, HD, 15; category 6, nonkeratinizing NPC, 86.

These EBV-associated disease conditions have been defined in our recent publications (20, 21). We further divided the NPC patients into different stages based on tumor size (T), lymph node involvement (N), the presence of hematogenous metastases (M), patient death or tumor remission, and AJC stages as described (22).

Radioimmunoprecipitation (RIPA) and Western blots

RIPA was conducted as described by us earlier (19). Briefly, 5 x 106 cells were labeled with 250 µCi [35S]methionine (NEN/DuPont), lysed in RIPA buffer in the presence of protease inhibitors, and immunoprecipitated using 2 µl of the undiluted ascites fluid of S12 Ab or 40 µl of the serum sample. The immunoprecipitates were resolved on 10% SDS-PAGE and detected by autoradiography. For Western blots, 5 x 106 cells were lysed in 50 mM Tris-HCl (pH 6.8), 2% SDS and sonicated for 15 s. The lysates were clarified by centrifugation (14,000 x g for 30 min at 4°C), and their protein concentrations were determined with a commercial kit (Bio-Rad Laboratories, Hercules, CA). Forty micrograms of the proteins were resolved on 10% SDS-PAGE, electroblotted onto nylon membranes (Immobilon, Millipore, Bedford, MA). The membranes were blocked in 5% skim milk powder in PBS and then incubated with appropriately diluted S12, CS1-4, or serum samples. After washings, blots were developed using alkaline phosphatase (AP)-conjugated secondary Abs (anti-mouse or anti-human IgG; both from Promega) and chromogenic substrates 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (Promega). For detection of LMP-1-specific IgA Abs, AP-conjugated rabbit anti-human IgA ({alpha}-chain specific; Dako; at 1:1000 dilution) was used in Western blots.

Indirect immunofluorescence

For this purpose, cells were fixed and premeabilized using the Cytofix/Cytoperm Kit (PharMingen, San Diego, CA). They were incubated with CS1-4 or S12 for 45 min at 4°C, washed, and stained with FITC-conjugated goat anti-mouse IgG. The stained cells were examined under fluorescent microscope or analyzed by flow cytometry using FACScan.

ELISA protocol

For the quantitation of anti-LMP-1 Abs, a LMP-1-specific ELISA was developed using a protocol described by us earlier (23) after some modifications. Briefly, plastic ELISA plates (Corning, Corning, NY; catalog number 28805) were coated (at 4°C overnight) with 50 µl of the cell lysates (protein content, 100 µg/ml) from LMP-1-expressing or control vector-transfected BJA-B cells. These lysates were obtained as described above for Western blots. The wells were washed three times with wash buffer containing 0.1% gelatin (Bio-Rad) and 0.05% Tween 20 in PBS and blocked with 0.2% gelatin in PBS (200 µl/well) at 4°C overnight. After washing, the wells were incubated with 50 µl of the serum dilution and incubated at room temperature for 2 h. After three subsequent washings with the wash buffer, 50 µl of 1:7500 diluted AP-conjugated anti-human IgG (heavy and light chain, Promega) was added. After incubation at room temperature for 2 h with the secondary Ab, the plates were washed extensively with the wash buffer, and colors were developed by using 50 µl p-nitrophenyl phosphate (Sigma, St. Louis, MO; catalog number N-9389, 1 mg/ml) dissolved in 0.1 M glycine buffer (pH 10.4) containing 1 mM MgCl2 and 1 mM ZnCl2. The reactions were stopped 30 min later with the addition of 50 µl of 3 N NaOH, and OD was measured at 405 {lambda} in an automatic ELISA reader (Easy Reader EAR 400AT, STL Labinstruments, Salzburg, Austria).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Detection of anti-LMP Abs by Western blots

The cell clones that we used as a source of LMP-1 in these studies could be easily stained and detected by flow cytometry using anti-LMP Abs (CS1–4 or S12) and FITC-conjugated secondary Abs after permeabilization (Fig. 1Go). These Abs, however, were unable to stain these cells by indirect membrane immunofluorescence without permeabilization (data not shown). With these Abs, LMP-1 could also be demonstrated in these cells by RIPA and Western blots (Fig. 2Go and data not shown). Following this, we used sera from EBV-seronegative and healthy EBV-seropositive individuals and from NPC patients in the RIPA and Western blots to see whether they contained anti-LMP Abs. As shown in Figs. 3Go and 4, several NPC sera showed positivity for these Abs. However, only a single sample of 20 EBV-seropositive healthy sera showed positivity for anti-LMP Abs. Sera from EBV-seronegative individuals were always negative both in RIPA and Western blots (data not shown). These data strongly suggested the presence of anti-LMP humoral responses in EBV-infected individuals. Interestingly, sera from CEI and IM patients were negative for these Abs. Table IGo depicts the percent positivity of sera for anti-LMP-1 Abs for various EBV-associated disease conditions. Within NPC patients, the percentage of anti-LMP Ab-positive sera showed a clear increasing trend in higher stages of the tumor (Table IIGo). An overwhelmingly high percentage of the dead NPC patients was positive for anti-LMP Abs (Table IIGo). In the case of HD, all EBER and/or LMP-positive tumors were positive for these Abs (Table IIIGo). One (of six) EBER-negative HD patient also contained these Abs. Taken together, these results illustrate the presence of anti-LMP humoral responses, especially in patients with EBV-associated NPC and HD.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 1. Immunofluorescence of LMP-expressing cells. Cells (1 x 106) were permeabilized and fixed using a Cytofix/Cytoperm Kit (Phar-Mingen) and then incubated with mAb CS1-4 (Dako, 5 µl/sample) or S12 (10 µl of 1:500 diluted ascites fluid per sample) for 45 min at room temperature. After extensive washings with PBS, the samples were incubated with 100 µl of 1:10 diluted FITC-conjugated goat anti-mouse 1gG (Becton Dickinson, Mountain View, CA) for a further 45 min. After three washes, the cells were examined by flow cytometry using FACscan. A compares the fluorescence profile of LMP-1-expressing cells with control vector-transfected cells when S12 mAb was used, whereas B depicts the same using CS1-4 mAb. In both panels, the y-axis denotes number of cells, and the x-axis depicts fluorescence intensity in arbitrary log units.

 


View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 2. Western blot of the LMP-expressing and control BJA-B cell clones. LMP-1 was detected on Western blots as described in Materials and Methods. Lanes 1 and 3 depict lysates from LMP-expressing cells, and lanes 2 and 4 depict lysates from the control cells. Lanes 1 and 2 were developed using CS1-4, and lanes 3 and 4 were developed using isotype-matched mouse Ab of an irrelevant specificity. LMP band is indicated by an arrow.

 


View larger version (70K):
[in this window]
[in a new window]
  		FIGURE 3. RIPA for detection of anti-LMP Abs in sera. LMP-1 was immunoprecipitated from the 35S-labeled cell lysates and resolved on 10% SDS-PAGE as described in Materials and Methods. The arrow shows the LMP band. A, Lanes 1–12 show immunoprecipitates using healthy EBV-seropositive sera. The same lanes in B represent immunoprecipitates after using NPC sera. Lanes 13 and 14 in both A and B show positive and negative controls, respectively, in which S12 or a control mouse Ab were used.

 

View this table:
[in this window]
[in a new window]
  		Table I. Positivity of sera of different groups of subjects for anti-LMP-1 Abs by Western blots1

 

View this table:
[in this window]
[in a new window]
  		Table II. Detection of anti-LMP-1 Abs in different NPC stages by Western blots1

 

View this table:
[in this window]
[in a new window]
  		Table III. Association of serum anti-LMP-1 Abs with EBV status of tumor samples in HD1

 
Detection of LMP-specific Abs by ELISA

Although anti-LMP Abs were detected by RIPA and/or Western blots, in many cases the revealed bands were faint, showed background darkness, and were not amenable to quantitative analyses. Therefore, to quantitate these Abs in the sera, we developed ELISA using lysates from LMP-expressing and control BJA-B cells. First, we determined the suitability of these lysates for ELISA using CS1-4 or S12 Abs. Both the LMP-1-specific Abs gave higher OD as compared with control Abs when lysates from LMP-1-expressing cells were used, whereas the OD of these Abs did not differ from those of control Abs when lysates from the control-transfected cells were used, suggesting specificity of the ELISA (data not shown). We then used pooled sera from EBV-seronegative subjects, EBV-seropositive healthy subjects, and from each EBV-associated disease separately in the ELISA. The results of the ELISA are depicted in Fig. 5Go. The highest ODs were observed in NPC sera followed by the sera from HD patients. The OD values from these two diseases were significantly (p <= 0.05) higher than those of HI, IM, and CEI sera at all dilutions tested (Fig. 5Go).



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 5. ELISA for anti-LMP-1 Abs in the sera of EBV-associated diseases. ELISA for anti-LMP Abs was performed using the indicated serum dilutions. All the sera in EBV-associated disease conditions were tested individually in the ELISA. The ELISA plates were coated with lysates from LMP-expressing BJA-B cells, and OD values were determined at 405 {lambda} as described in Materials and Methods. Note the higher average OD values at each dilution for NPC and HD sera than for CEI, HI, or IM sera.

 
Anti-LMP-1 Abs in the sera of NPC patients with different stages of the tumor

The NPC sera were classified as described in Materials and Methods. To determine whether these anti-LMP-1 Abs differed in various stages of this tumor, all sera from each category were tested individually in ELISA and average OD values for each category of serum were calculated. As shown in Fig. 6Go, A–D, in each category, a trend toward higher average OD with increase in tumor size (T0->T4), increased nodal involvement (N0->N3) and with metastasis is evident. Higher average ODs were also seen in sera from dead patients as compared with the patients with remissions. Furthermore, these OD values also increased in advanced stages of the tumor. These results strongly suggest a prognostic value of these Abs.



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 6. ELISA for anti-LMP-1 Abs in different NPC stages. The NPC sera (86) were classified into different stages depending on tumor size, nodal involvement, metastasis, deaths, and tumor remissions as described in Materials and Methods. Shown are average OD values for different stages of tumor size (A), presence or absence of metastases (B), nodal involvement (C), and patient deaths or tumor remissions (D). A, B, and C in A represent NPC stages as indicated in Table VGo. A clear trend of increase in OD values at all the serum dilutions in advanced stages of the tumor is evident.

 
Detection of LMP-1-specific IgA in NPC sera

Because earlier studies from this and other laboratories have shown that Abs of IgA isotype against several EBV Ags are of diagnostic and prognostic value, especially for NPC (16, 20, 24), we did Western blots using AP-conjugated human {alpha}-chain-specific secondary Abs to detect these Abs in these sera. Fig. 7Go depicts a typical Western blot, and the results from several blots are shown in Table IVGo. Only NPC sera (50%) were found to be positive for anti-LMP-1 IgA Abs. The percent positivity for this Ab in NPC sera also tended to increase in advanced stages of the tumor (Table VGo). These data collectively show that anti-LMP IgA Abs are pathognomonic for NPC and have prognostic value.



View larger version (95K):
[in this window]
[in a new window]
  		FIGURE 7. Western blots for detection of anti-LMP IgA Abs in sera. The Western blots from the LMP-expressing and control cells were cut into small strips, and each strip was incubated with 1:500 diluted serum sample (at room temperature for 2 h), washed, and then incubated with AP-conjugated anti-human IgA ({alpha}-chain-specific) Ab and the color was developed with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (Promega). The arrow shows the LMP protein location. Lanes 1–5 were developed with sera from healthy EBV-seropositive individuals, and lanes 6–10 with NPC sera.

 

View this table:
[in this window]
[in a new window]
  		Table IV. Positivity of sera for anti-LMP-1-specific IgA for different EBV-associated conditions by Western blots1

 

View this table:
[in this window]
[in a new window]
  		Table V. Percent positivity of sera for anti-LMP-1-specific IgA in different stages of NPC by Western blots1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
LMP is an EBV-encoded oncoprotein which is expressed very frequently in several EBV-associated malignancies. In these malignancies, the human host mounts strong anti-EBV cellular and humoral immune responses. These humoral (Ab) responses against several EBV Ags, e.g., early Ags, viral capsid Ags (VCA), have been used as important tools in the diagnosis and prognosis of these EBV-associated diseases. Although LMP is thought to play an important role in EBV-associated malignancies and LMP-specific CTL responses have been well documented in EBV-infected individuals, quantitative studies of anti-LMP Ab responses and their significance in EBV-associated diseases have not yet been reported. To our knowledge, this is the first report to quantify these Abs by ELISA using lysates from LMP-expressing, EBV genome-negative cells. Earlier, Modrow and Wolf (25) and Dillner et al. (26) suggested the presence of these Abs in human sera by using synthetic peptides corresponding to LMP. Rowe et al. (27) and Chen et al. (28) used recombinant LMP or cell lysates from an EBV-immortalized human cord blood cell line IB4 and demonstrated the presence of anti-LMP-1 Abs in the sera of healthy EBV-seropositive and NPC patients by immunoprecipitation and immunoblotting. These methods, however, are not suitable for quantification of Abs in the sera. We developed ELISA to quantitatively compare anti-LMP Abs in the sera of different EBV-associated diseases and, for this purpose, used lysates from cells that expressed high levels of LMP after transfection with an LMP expression vector. The use of an EBV genome-negative BL cell line BJA-B (developed by J.M. in the mid-1970s; 18) ensured that we were not detecting Abs against other EBV Ags in our ELISA. Recently, Meji et al. (29) reported that EBV-transformed B cells expressed little LMP and were not suitable for detecting anti-LMP Abs in human sera. They used recombinant LMP produced in insect cells in their ELISA for this purpose and found very low positivity of various human sera for anti-LMP Abs; e.g., they found only 7.5% NPC sera positive for these Abs as compared with 70.9% reported in the present study. This low positivity of sera in their assays may be due to heavy glycosylation and the consequent masking of immunoreactive epitopes on LMP. The proteins produced in insect cells with the use of recombinant baculoviruses are known to be heavily glycosylated (30).

Although the presence of anti-LMP Abs in normal sera was reported earlier (27), our results show that only a fraction (1 in 20; 5%) of healthy EBV-seropositive individuals are positive for these Abs. Concerning HD, we found one patient positive for anti-LMP Abs despite the absence of EBV genome in its tumor (Reed-Sternberg) cells. This confirms an earlier report (31) that, unlike Burkitt’s lymphoma, the presence or absence of anti-EBV Abs is not predictive of the EBV status of the tumor cells in HD. Interestingly, sera from IM and CEI patients were negative for these Abs. More importantly, two-thirds or more sera from patients with EBV-associated malignancies (NPC and HD) were positive for these Abs. Furthermore, the OD values of these Abs in ELISA also correlated positively with advanced stages of the tumor in NPC patients. IgA Ab titers for several EBV Ags, e.g., early Ag, viral capsid Ag, gp350/220, have been documented to have both diagnostic and prognostic significance in NPC (2, 24). We also found that anti-LMP Abs of IgA isotype were detectable only in the sera of NPC patients, suggesting pathognomonicity of these Abs to this malignant condition. Collectively, these results show the potential significance of anti-LMP Abs for the diagnosis and prognosis of EBV-associated malignancies.

The LMP-specific mAbs CS1-4 and S12 are known to recognize intracellular parts of LMP (32, 33), and LMP Ab-positive sera stained our LMP-expressing cells in indirect immunofluorescence assays only when these cells were first permeabilized. These Abs and sera, however, were unable to stain these cells without prior permeabilization, suggesting that these Abs recognized only intracellular parts of the protein. Similar observations were made earlier by Rowe et al. (27). These data suggest that extracellular parts of LMP (the three 8- to 12-amino acid-long turns) are not immunogenic and are not targeted by human anti-LMP Abs. This is further supported buy our observations that LMP-expressing cells are not killed by NK cells in Ab-dependent cellular cytotoxicity (ADCC) assays as described by us for anti-gp350/220 Abs (Ref. 20 and data not shown). Thus, it appears that anti-LMP humoral response does not play a role in the control of EBV-infected/immortalized cells. Therefore, LMP-expressing malignant cells are not under pressure to accumulate mutations to evade this response. This also reinforces the commonly held notion that anti-EBV cellular immune responses are more important to control EBV infections (16, 17).

LMP is a transforming protein that mimics (but is not identical with) a constitutively activated CD40 receptor (1, 3, 7, 9, 10, 14). It activates several cellular genes, acts as a classical oncogene and is the frequently expressed EBV protein in several EBV-associated malignancies. The expression of LMP-1 in 100% cases of rarely detected preinvasive NPC lesions, and monoclonality of EBV genomes in these tumors strongly suggest a causative role of EBV and this EBV protein in this tumor (34). LMP inhibits differentiation of epithelial cells and induces the expression of several antiapoptotic genes, e.g., bcl-2, A20, Mcl-1, depending on the cell type (3, 35). Antisense oligos to the first five codons of LMP induce apoptosis in EBV-immortalized B cells (36). Taken together, these observations strongly suggest a role for LMP in EBV-induced tumorigenicity. However, there is no clear in vivo evidence for such a role for this protein. On the contrary, LMP per se is cytostatic or even cytotoxic for human cells (37, 38), and LMP-positive tumors have better prognosis than LMP-negative tumors (39, 40). LMP is targeted by EBV-specific CTL and its expression in Burkitt’s lymphoma makes them susceptible to lysis by these CTL (39). It also enhances expression of MHC class I Ags and costimulatory molecules, e.g., CD80, CD86, and consequently increases the presentation of endogenous and exogenous Ags to the immune system (41, 42, 43). LMP-1-expressing Burkitt’s lymphomas are less tumorigenic than their LMP-negative counterparts in nude and SCID mice (39). Recently, Cherney et al. (44) demonstrated that expression of LMP augments nonspecific antitumor immunity by inducing the secretion of cytokines and chemokines. The EBV-induced tumors, however, may have evolved strategies to overcome these responses by accumulating mutations in LMP. An LMP gene cloned from an NPC patient was reported to be more tumorigenic and less immunogenic in mice than its B95-8 counterpart (45). Mutations in the promoter region and in the C-terminal cytoplasmic region of LMP have been documented by several workers (46, 47, 48, 49). A characteristic 30-bp mutation in the C-terminal part has attracted much attention (49, 50, 51). However, there is not yet convincing in vivo evidence of the role of this or any other LMP mutation in increased tumorigenicity. Our results suggest that anti-LMP Abs recognize hidden intracellular parts of LMP and do not mediate ADCC and therefore may not be driving the accumulation of these mutations in EBV-associated tumors. Instead, these mutations may enable tumors to escape LMP-specific CTL, render this protein unable to stimulate nonspecific antitumor immunity, or reduce its cytotoxic/cytostatic effects. Because human anti-LMP-1 Abs recognized only intracellularly located parts of the protein, the immune system may be exposed to LMP-1 on lysis of LMP-expressing EBV-infected cells and/or by virolysis of released virions. The titers of anti LMP Abs, therefore, may reflect the magnitude of this exposure. Our data also suggest that anti-LMP Abs increase in advanced stages of NPC. The increase in anti-LMP Abs in advanced NPC stages may simply reflect an enhanced exposure to LMP in these patients. Alternatively, these patients may be mounting disproportionally higher humoral responses as compared with the CTL responses. Because we have not determined LMP-specific cellular immune responses in these patients, further studies will be needed to address these issues.

This quantitation and analysis of anti-LMP Abs using ELISA underscores the potential usefulness of these Abs in the diagnosis and prognosis of EBV-associated malignancies, particularly NPC.



View larger version (141K):
[in this window]
[in a new window]
  		FIGURE 4. Western blot analysis for detection of anti-LMP Abs in sera. The Western blots from LMP-1-expressing and control cells were cut into strips. The strips were developed using human sera and AP-conjugated goat anti-human IgG as detailed in Materials and Methods. Arrow, LMP location. The sera used in developing these strips were healthy seropositives (lanes 1–3), infectious mononucleosis (lanes 4–6), CEI (lanes 7–9), HD (lanes 10 and 11), and NPC (lanes 12–14). Lanes 15 and 16 represent positive (S12) and negative (control Ab) controls, respectively.

 

   Acknowledgments
 
We thank Dr. Nancy Raab-Traub for the LMP-1 expression plasmid, Elliot Kieff for S12 Abs, Mary Blagdon for FACS analysis, and Micheline Patenaude for secretarial assistance.


   Footnotes
 
1 This work was supported by the Medical Research Council of Canada and Associazione Italiana per la Ricerca sul Cancero. A.A. is a recipient of the Medical Research Council of Canada Scholarship. Back

2 Address correspondence and reprint requests to Dr. Ali Ahmad, Laboratory of Immunovirology, Ste.-Justine Hospital, 3175 Côte Ste.-Catherine, Montreal, Quebec, H3T 1C5, Canada. E-mail address: Back

3 Abbreviations used in this paper: IM, infectious mononucleosis; CEI, chronic EBV infection; LMP-1, latent membrane protein-1; NPC, nasopharyngeal carcinoma; EBER, EBV RNA; HI, healthy EBV seropositives; RIPA, radioimmunoprecipitation assay; AP, alkaline phosphatase; ADCC, Ab-dependent cellular cytotoxicy. Back

Received for publication November 2, 1999. Accepted for publication December 17, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kieff, E.. 1996. Epstein-Barr virus and its replication. ed. Fields Virology 2343.-2396. Lippincott-Raven, Philadelphia.
  2. Rickinson, A. B., E. Kieff. 1996. Epstein-Barr virus. ed. Fields Virology 2397.-2446. Lippincott-Raven, Philadelphia.
  3. Liebowitz, D.. 1994. Nasopharyngeal carcinoma: the Epstein-Barr virus association. Semin. Oncol. 21:376.[Medline]
  4. Henderson, S., M. Rowe, C. Gregory, D. Croom-Carter, F. Wang, R. Longnecker, E. Kieff, A. Rickinson. 1991. Induction of Bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death. Cell 65:1107.[Medline]
  5. Erickson, K. D., J. M. Martin. 1997. Early detection of the lytic LMP-1 protein in EBV-infected B cells suggests its presence in the virion. Virology 284:1.
  6. Eliopoulos, A. G., A. B. Rickinson. 1998. Epstein-Barr virus: LMP1 masquarades as an active receptor. Curr. Biol. 8:R196.[Medline]
  7. Eliopoulos, A. G., S. M. Blake, J. E. Floettmann, M. Rowe, L. S. Young. 1999. Epstein-Barr virus encoded latent membrane protein 1 activates the JNK pathway through its extreme C terminus via a mechanism involving TRADD and TRAF 2. J. Virol. 73:1023.[Abstract/Free Full Text]
  8. Miller, W. E., G. Mosialos, E. Kieff, N. Raab-Traub. 1997. Epstein-Barr virus LMP1 induction of the epidermal growth factor receptor is mediated through a TRAF signaling pathway distinct from NF-{kappa}B activation. J. Virol. 71:586.[Abstract]
  9. Mosialos, G., M. Birkenbach, R. Yalamanchili, T. vanArsdale, C. Ware, E. Kieff. 1995. The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family. Cell 80:389.[Medline]
  10. Devergne, O., E. Hatzivassiliou, K. M. Izumi, K. M. Kaye, M. F. Kleijnen, E. Kieff, G. Mosialos. 1996. Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 domain important for B-lymphocyte transformation: role in NF-{kappa}B activation. Mol. Cell. Biol. 16:7098.[Abstract]
  11. Sandberg, M., W. Hammerschmidt, B. Sugden. 1997. Characterization of LMP-1’s association with TRAF1, TRAF2, and TRAF3. J. Virol. 71:4649.[Abstract]
  12. Kaye, K. M., K. M. Izumi, E. Kieff. 1993. Epstein-Barr virus latent membrane protein 1 is essential for B lymphocyte transformation. Proc. Natl. Acad. Sci. USA 90:9150.[Abstract/Free Full Text]
  13. Wang, F., C. Gregory, C. Sample, M. Rowe, D. Liebowitz, R. Murray, A. Rickinson, E. Kieff. 1990. Epstein-Barr virus latent-membrane protein (LMP1) and nuclear proteins 2 and 3C are effectors of phenotypic changes in B lymphocytes: EBNA-2 and LMP1 cooperatively induce CD23. J. Virol. 64:2309.[Abstract/Free Full Text]
  14. Rowe, M., M. Peng-Pilon, D. S. Huen. 1994. Upregulation of bcl-2 by the Epstein-Barr virus latent membrane protein LMP1: a B cell-specific response that is delayed relative to NF-{kappa}B activation and to induction of cell surface markers. J. Virol. 68:5602.[Abstract/Free Full Text]
  15. Arvanitakis, L., N. Yaseen, S. Sharma. 1995. Latent-membrane protein-1 induces cyclin D2 expression, pRb hyperphosphorylation, and loss of TGF-ß1-mediated growth inhibition in EBV-positive B cells. J. Immunol. 155:1047.[Abstract]
  16. Khanna, R., S. R. Burrows, D. J. Moss. 1995. Immune regulation in Epstein-Barr virus-associated diseases. Microbiol. Rev. 59:387.[Abstract/Free Full Text]
  17. Rickinson, A. B., J. D. Moss. 1997. Human cytotoxic T lymphocyte responses to Epstein-Barr virus infection. Annu. Rev. Immunol. 15:405.[Medline]
  18. Menezes, J., W. Leibold, G. Klein, G. Clements. 1975. Establishment and characterization of an Epstein-Barr virus (EBV)-negative lymphoblastoid cell line (BJA-B) from an exceptional, EBV-genome-negative African Burkitt lymphoma. Biomedicine 22:276.[Medline]
  19. Ahmad, A., A. Ladha, E. A. Cohen, J. Menezes. 1993. Stable expression of the transfected HIV-1 env gene in a human B cell line: characterization of gp120-expressing clones and immunobiological studies. Virology 192:447.[Medline]
  20. Xu, J., A. Ahmad, M. Blagdon, M. D’Addario, J. F. Jones, R. Dolcetti, E. Vaccher, U. Prasad, J. Menezes. 1998. The Epstein-Barr virus (EBV) major envelope glycoprotein gp350/220-specific antibody reactivities in the sera of patients with different EBV-associated diseases. Int. J. Cancer 79:481.[Medline]
  21. Xu, J., J. Menezes, U. Prasad, A. Ahmad. 1999. Elevated serum levels of transforming growth factor ß1 in the Epstein-Barr virus-associated nasopharyngeal carcinoma patients. Int. J. Cancer 84:396.[Medline]
  22. Teo, P. M. L., D. N. Leibowitz, R. R. A. Weichselbaum. 1991. Proposed modification of the Ho stage-classification for nasopharyngeal carcinoma. Radiol. Oncol. 21:11.
  23. Tanner, J. E., M. X. Wei, C. Alfieri C, A. Ahmad, P. Tailor, T. Ooka, J. Menezes. 1997. Antibody and antibody-dependent cellular cytotoxicity (ADCC) responses against the BamHIA rightward open reading frame-1 (BARF-1) protein of Epstein-Barr virus (EBV) in EBV-associated disorders. J. Infect. Dis. 175:38.[Medline]
  24. Xu, J. W., A. Ahmad, U. Prasad, J. Menezes. 1998. Clinical relevance of IgA antibodies to the Epstein-Barr virus envelope glycoprotein gp350 in the sera of nasopharyngeal carcinoma patients. J. Clin. Microbiol. 36:3740.[Free Full Text]
  25. Modrow, S., H. Wolf. 1986. Characterization of two related Epstein-Barr virus-encoded membrane proteins that are differentially expressed in Burkitt’s lymphoma and in vitro transformed cell lines. Proc. Natl. Acad. Sci. USA 83:5703.[Abstract/Free Full Text]
  26. Dillner, J., R. Szigeti, W. Henle, R. A. Lerner, G. Klein. 1987. Cellular and humoral immune responses to synthetic peptides deduced from the amino-acid sequences of Epstein-Barr virus-encoded proteins in EBV-transformed cells. Int. J. Cancer 40:455.[Medline]
  27. Rowe, M., J. Finke, R. Szigeti, G. Klein. 1988. Characterization of the serological response in man to the latent membrane protein and the six nuclear antigens encoded by Epstein-Barr virus. J. Gen. Virol. 69:1217.[Abstract/Free Full Text]
  28. Chen, H.-F., S. Kevan-Jah, K.-O. Suentzenich, F. A. Grasser, N. Muller-Lantzsch. 1992. Expression of the Epstein-Barr virus latent membrane protein (LMP) in insect cells and detection of antibodies in human sera against this protein. Virology 190:106.[Medline]
  29. Meji, P., M. B. H. J. Vervoort, J. Aarbiou, P. van Dissel, A. Brink, E. Bloemena, C. J. L. M. Meijer, J. M. Middledrop. 1999. Restricted low-level human antibody responses against Epstein-Barr virus (EBV)-encoded latent membrane protein 1 in a subgroup of patients with EBV-associated diseases. J. Inf. Dis. 179:1108.[Medline]
  30. Smith, G. E., M. D. Summers, M. J. Fraser. 1987. Production of human ß-interferon in insect cells infected with a baculovirus expression vector. Mol. Cell. Biol. 3:2156.
  31. Levine, P. H., G. Pallesen, P. Ebbesen, N. Harris, A. S. Evans, N. Mueller. 1994. Evaluation of Epstein-Barr virus antibody patterns and detection of viral markers in the biopsies of patients with Hodgkin’s disease. Int. J. Cancer 59:48.[Medline]
  32. Rowe, M., H. S. Evans, L. S. Young, K. Hennessy, E. Kieff, A. B. Rickinson. 1987. Monoclonal antibodies to the latent membrane protein of Epstein-Barr virus reveal heterogeneity of the protein and inducible expression in virus-transformed cells. J. Gen. Virol. 68:1575.[Abstract/Free Full Text]
  33. Mann, K. P., D. Staunton, D. A. Thorley-Lawson. 1985. Epstein-Barr virus-encoded protein found in plasma membranes of transformed cells. J. Virol. 55:710.[Abstract/Free Full Text]
  34. Pathmanathan, R., U. Prasad, R. Sadler, K. Flynn, N. Raab-Traub. 1995. Clonal proliferation of cells infected with Epstein-Barr virus in pre-invasive lesions related to nasopharyngeal carcinoma. N. Engl. J. Med. 333:693.[Abstract/Free Full Text]
  35. Dawson, C. W., A. B. Rickinson, L. S. Young. 1990. Epstein-Barr virus latent membrane protein inhibits human epithelial cell differentiation. Nature 344:777.[Medline]
  36. Kenney, J. L., M. E. Guinness, T. Curiel, J. Lacy. 1998. Antisense to the Epstein-Barr virus (EBV)-encoded latent membrane protein 1 in EBV-immortalized B cells. Blood 92:1721.[Abstract/Free Full Text]
  37. Floettmann, J. E., K. Ward, A. B. Rickinson, M. Row. 1996. Cytostatic effect of Epstein-Barr virus latent membrane protein-1 analyzed using tetracycline-related expression in B cell lines. Virology. 223:29.[Medline]
  38. Hammerschmidt, M., B. Sudgen, V. R. Baichwal. 1989. The transformation domain alone of the latent membrane protein of Epstein-Barr virus is toxic to cells when expressed at high levels. J. Virol. 63:2469.[Abstract/Free Full Text]
  39. Cuomo, L., T. Ramquist, P. Trivedi, F. Wang, G. Klein, M. G. Masucci. 1992. Expression of the Epstein-Barr virus (EBV)-encoded membrane protein LMP1 impairs the in vitro growth, clonability and tumorigenicity of an EBV-negative Burkitt lymphoma line. Int. J. Cancer 51:949.[Medline]
  40. Hu, L.-F., F. Chen, Q-F. Zhen, Y. W. Zhang, Y. Luo, X. Zheng, G. Winberg, I. Ernberg, G. Klein. 1995. Differences in the growth pattern and clinical course of EBV-LMP1 expressing and non-expressing nasopharyngeal carcinomas. Eur. J. Cancer 31A:658.
  41. Cuomo, L., P. Trivedi, F. Wang, G. Wimberg, G. Klein, M. Masucci. 1990. Expression of the Epstein-Barr virus (EBV)-encoded membrane antigen LMP increases the stimulatory capacity of EBV negative B lymphoma lines in allogeneic mixed lymphocyte cultures. Eur. J. Immunol. 20:2293.[Medline]
  42. Agathanggelou, A., G. Niedobitek, R. Chen, J. Nicholls, W. Yin, L. S. Young. 1995. Expression of immune regulatory molecules in Epstein-Barr virus-associated nasopharyngeal carcinomas with prominent lymphoid stroma: evidence for a functional interaction between epithelial tumor cells and infiltrating lymphoid cells. Am. J. Pathol. 147:1152.[Abstract]
  43. Murray, P. G., C. M. Constandinou, J. Crocker, L. S. Young, R. F. Ambinder. 1998. Analysis of major histocompatibility complex class I, TAP expression, and LMP2 epitope sequence in Epstein-Barr virus-positive Hodgkin’s disease. Blood 92:2477.[Abstract/Free Full Text]
  44. Cherney, B. W., C. Sgadari, C. Kanegane, F. Wang, G. Tosato. 1998. Expression of the Epstein-Barr virus protein LMP1 mediated tumor regression in vivo. Blood. 91:2491.[Abstract/Free Full Text]
  45. Trividi, P., L. F. Hu, F. Chen, B. Christensson, M. G. Masucci, G. Klein, G. Winberg. 1994. Epstein-Barr virus (EBV)-encoded membrane protein LMP1 from a nasopharyngeal carcinoma is non-immunogenic in a murine model system, in contrast to a B cell-derived homologue. Eur. J. Cancer 30A:84.
  46. Knecht, H., E. Buchmann, P. Brousset, S. Rothenberger, H. Einsek, V. S. Lestou, G. Delsol, F. Buchmann, P. F. Ambros, B. F. Odermatt. 1995. Mutational hot spots within the carboxy terminal region of the LMP1 oncogene of Epstein-Barr virus are frequent in lymphoproliferative disorders. Oncogene 10:523.[Medline]
  47. Dolcetti, R., P. Zancai, V. D. Re, A. Gloghini, B. Bigoni, B. Pivetta, S. D. Vita, A. Carbone, M. Boiocchi. 1997. Epstein-Barr virus strains with latent membrane protein-1 deletions: prevalence in the Italian population and high association with human immunodeficiency virus-related Hodgkin’s disease. Blood 89:1723.[Abstract/Free Full Text]
  48. Sandvej, K., J. W. Gratama, M. Munch, X.-G. Zhou, R. L. H. Bolhuis, B. S. Andersen, N. Gregersen, S. Hamilton-Dutoit. 1997. Sequence analysis of the Epstein-Barr virus (EBV) latent membrane protein-1 gene and promoter region: identification of four variants among wild-type EBV isolates. Blood 90:323.[Abstract/Free Full Text]
  49. Hu, L. F., E. R. Zabarovky, F. Chen, S. L. Cao, I. Ernberg, G. Klein, G. Winberg. 1991. Isolation and sequencing of the Epstein-Barr virus BNLF-1 gene (LMP-1) from a Chinese nasopharyngeal carcinoma. J. Gen. Virol. 72:2399.[Abstract/Free Full Text]
  50. Chen, M. L., C. N. Tsai, C. L. Liang, C. S. Shu, C. R. Huang, D. Sulitzeanu, S. T. Liu, Y. S. Chang. 1992. Cloning and characterization of the latent membrane protein (LMP) of a specific Epstein-Barr virus variant derived from the nasopharyngeal carcinoma in the Taiwanese population. Oncogene 7:2131.[Medline]
  51. Li, S-N., Y-S. Chang, S-T. Liu. 1996. Effect of a 10-amino acid deletion of the oncogenic activity of latent membrane protein 1 of Epstein-Barr virus. Oncogene 12:2129.[Medline]



This article has been cited by other articles:


Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
W.-L. Hsu, J.-Y. Chen, Y.-C. Chien, M.-Y. Liu, S.-L. You, M.-M. Hsu, C.-S. Yang, and C.-J. Chen
Independent Effect of EBV and Cigarette Smoking on Nasopharyngeal Carcinoma: A 20-Year Follow-Up Study on 9,622 Males without Family History in Taiwan
Cancer Epidemiol. Biomarkers Prev., April 1, 2009; 18(4): 1218 - 1226.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. Kobayashi, T. Nagato, M. Takahara, K. Sato, S. Kimura, N. Aoki, M. Azumi, M. Tateno, Y. Harabuchi, and E. Celis
Induction of EBV-Latent Membrane Protein 1-Specific MHC Class II-Restricted T-Cell Responses against Natural Killer Lymphoma Cells
Cancer Res., February 1, 2008; 68(3): 901 - 908.
[Abstract] [Full Text] [PDF]

Home page
 CVIHome page
R. Tedeschi, E. Pin, D. Martorelli, E. Bidoli, A. Marus, C. Pratesi, M. T. Bortolin, S. Zanussi, E. Vaccher, R. Dolcetti, et al.
Serum Antibody Response to Lytic and Latent Epstein-Barr Virus Antigens in Undifferentiated Nasopharyngeal Carcinoma Patients from an Area of Nonendemicity
Clin. Vaccine Immunol., April 1, 2007; 14(4): 435 - 441.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C.-L. Tsai, H.-P. Li, Y.-J. Lu, C. Hsueh, Y. Liang, C.-L. Chen, S. W. Tsao, K.-P. Tse, J.-S. Yu, and Y.-S. Chang
Activation of DNA Methyltransferase 1 by EBV LMP1 Involves c-Jun NH2-Terminal Kinase Signaling
Cancer Res., December 15, 2006; 66(24): 11668 - 11676.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
R. Ahmad, S. T. A. Sindhu, E. Toma, R. Morisset, and A. Ahmad
Elevated Levels of Circulating Interleukin-18 in Human Immunodeficiency Virus-Infected Individuals: Role of Peripheral Blood Mononuclear Cells and Implications for AIDS Pathogenesis
J. Virol., November 13, 2002; 76(24): 12448 - 12456.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
J. Xu, A. Ahmad, and J. Menezes
Preferential Localization of the Epstein-Barr Virus (EBV) Oncoprotein LMP-1 to Nuclei in Human T Cells: Implications for Its Role in the Development of EBV Genome-Positive T-Cell Lymphomas
J. Virol., March 19, 2002; 76(8): 4080 - 4086.
[Abstract] [Full Text] [PDF]

Home page
 NeurologyHome page
J. M. Woulfe, D. G. Munoz, J. De Keyser, and M. Lousa
Status epilepticus with neuron-reactive serum antibodies: Response to plasma exchange
Neurology, November 14, 2000; 55(9): 1421 - 1421.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xu, J.
Right arrow Articles by Menezes, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xu, J.
Right arrow Articles by Menezes, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS