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 Donati, D.
Right arrow Articles by Bejarano, M. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Donati, D.
Right arrow Articles by Bejarano, M. T.
The Journal of Immunology, 2006, 177: 3035-3044.
Copyright © 2006 by The American Association of Immunologists, Inc.

Increased B Cell Survival and Preferential Activation of the Memory Compartment by a Malaria Polyclonal B Cell Activator1

Daria Donati2,{dagger}, Bobo Mok*, Arnaud Chêne*,{dagger}, Hong Xu{dagger}, Mathula Thangarajh{ddagger}, Rickard Glas{dagger}, Qijun Chen§, Mats Wahlgren* and Maria Teresa Bejarano*,{dagger}

* Microbiology and Tumorbiology Center, Karolinska Institutet, {dagger} Center for Infectious Medicine, Department of Medicine, {ddagger} Division of Neurology R54, Karolinska Institutet, Karolinska University Hospital Huddinge, and § Swedish Institute for Infection Disease Control, Stockholm, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Chronic malaria infection is characterized by polyclonal B cell activation, hyperglobulinemia, and elevated titers of autoantibodies. We have recently identified the cysteine-rich interdomain region 1{alpha} (CIDR1{alpha}) of the Plasmodium falciparum erythrocyte membrane protein 1 as a T cell-independent polyclonal B cell activator and Ig binding protein. Here, we show that, although the binding affinity of CIDR1{alpha} to human IgM and IgG is relatively low, B cell activation still proceeds. CIDR1{alpha} rescues tonsillar B cells from apoptosis, and increases the proportion of cycling cells. Comparison of the impact on naive and memory B cell compartment indicated that CIDR1{alpha} preferentially activates memory B lymphocytes. Analysis of the gene expression profiles induced by CIDR1{alpha} and anti-Ig activation using a cDNA microarray demonstrated a low degree of homology in the signatures imposed by both stimuli. The microarray data correlate with the functional analysis demonstrating that CIDR1{alpha} activates various immunological pathways and protects B cells from apoptosis. Together, the results provide evidence for a role of malaria in preferentially activating the memory B cell compartment. The polyclonal B cell activation and augmented survival induced by CIDR1{alpha} is of relevance for understanding the mechanisms behind the increased risk of Burkitt’s lymphoma in malaria endemic areas.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Aberrant immune activation induced by chronic infections with Plasmodium falciparum leads to polyclonal B cell activation characterized by the presence of hyperglobulinemia (1), elevated titers of autoantibodies (2, 3), and frequent occurrence of Burkitt’s lymphoma (4) and splenic lymphoma (5). The mechanisms that lead to this polyclonal B cell activation are poorly understood.

The marked effect of malaria infection on B cells is related both to the biology of the infection, and to the nature of the malarial Ags. P. falciparum-infected erythrocytes (IE)3 have the potential to directly interact with B cells in different anatomical sites and to induce B cell proliferation and differentiation into Ab-secreting cells. We have shown that a large proportion (83%) of fresh isolates of IE bind nonimmune Igs (6), suggesting that in the peripheral blood IE could interact with B cells through their surface Igs. Moreover, bloodborne Ags (and thus malarial Ags related to the erythrocytic phase) are trapped in the spleen where B cells represent ~40% of the splenocytes. Thus, IE and their constituent Ags could interact in the spleen with B cells displaying a variety of surface phenotypes, Ag-binding repertoires and signaling profiles (7). Among malarial Ags, the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of proteins often display Ig binding properties (8, 9). The Ig binding activity of the PfEMP1, cloned from two different P. falciparum strains, resides in two different variable domains, the Duffy binding-like domain 2beta (DBL2beta) and the cysteine-rich interdomain region 1{alpha} (CIDR1{alpha}) (8). The latter domain has been identified as a polyclonal B cell activator and an Ig binding protein (IBP) (10) with a binding pattern similar to that of another microbial IBP, the protein A of Staphylococcus aureus (8, 10, 11). Microbial IBPs are produced by protozoa, viruses, and bacteria (12), and play important physiological roles (13). During an infectious process, IBPs may act as an evasion mechanism to divert specific Ab responses (14, 15). CIDR1{alpha} binds to and activates purified B lymphocytes in vitro, an interaction partially mediated through the binding to surface Ig (10).

To further understand the impact of CIDR1{alpha} on the immune system, we analyzed its effect on the dynamics of the B cell compartment and compared the gene expression profiles during activation induced by CIDR1{alpha} and the triggering of the BCR via anti-Ig. The data show that CIDR1{alpha} preferentially induces the activation of the memory B cell compartment and that this activation seems to be different from the one imposed by anti-Ig treatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Production of recombinant Ags

CIDR1{alpha}, of the cloned strain FCR3S1.2var1, was cloned in the pGEX-4T plasmid (Amersham Biosciences) and expressed in Escherichia coli (BL21) as previously described (8). The CIDR1{alpha}-GST fusion-protein, referred to as CIDR1{alpha}, was expressed and purified according to the instructions of the manufacturer. GST produced by the empty vector was used as control. Henceforth, this is referred to as GST. The purity was determined by SDS-PAGE and Western blot as described (16) (data not shown).

B cells and cell culture

Buffy coats from blood of healthy individuals, never exposed to malaria, were obtained from the blood bank of the Karolinska Hospital. Mononuclear cells were isolated by centrifugation over Lymphoprep (Nycomed Pharma). CD19+ B cells were isolated by positive selection using the MACS cell separation system (Miltenyi Biotec) according to the manufacturer’s instructions; the B cell purity varied between 94 and 99%.

Tonsils were obtained from patients undergoing routine tonsillectomy at the Karolinska University Hospital. Lymphocyte suspensions were prepared by mincing the tissues and suspending the cells in complete RPMI 1640. Isolated mononuclear cells were depleted of T cells by two rounds of rosette formation with amino ethyl isothiouronium bromide-treated SRBC on ice. Rosettes were removed by centrifugation over Lymphoprep (17). The tonsillary B cell purity was >95% as revealed by FACS analysis after staining with the pan-T cell Ab (CD3) and the monocyte marker CD14. Cultures were maintained in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% FCS, 100 U/ml penicillin, and 2 mM glutamine. Purified B cells were cultured either in medium alone, or medium containing GST, CIDR1{alpha}, or anti-Ig F(ab')2 (10 µg/ml) (Jackson ImmunoResearch Laboratories), and incubated up to 48 h at 37°C and 5% CO2. Unless otherwise specified, the final concentration of GST and CIDR1{alpha} was 100 µg/ml.

Approval for these studies was obtained from the Karolinska Institutet Ethical Committee.

Phenotypic analysis

Saturating concentrations of PE- or avidin-conjugated monoclonal anti-human Abs (mAb) (BD Biosciences) were added to cell pellets collected after 48 h of culture and incubated for 30 min at 4°C. PE- and FITC-conjugated isotype-matched mAbs (BD Biosciences) with irrelevant specificities were used as negative controls. Fluorescence intensity was measured with a FACSCalibur flow cytometer, and analyzed using the CellQuest software (BD Biosciences).

B cell survival and cell cycle analysis

DNA profiles were obtained by staining cells with propidium iodide (PI) (Sigma-Aldrich). Cultured tonsillar B lymphocytes (0.5 x 106/ml) were washed with ice-cold PBS and resuspended in 100 µl of PBS containing PI (50 µg/ml), and 0.1% (v/v) Triton X-100. Cells were incubated for 6–8 h at 4°C before being analyzed on a FACSCalibur. The rescue from cell death was calculated from the percentage of cell death in different cultures, using the following formula: percentage of rescue = 1 – (percentage of cell death in CIDR1 cultures/percentage of cell death in control cultures) x 100.

The distribution of the cells in the sub G0-G1 fraction and in the different phases of the cell cycle was analyzed by the CellQuest software (BD Biosciences).

Immunoblot analysis

Lysates of B lymphocytes were separated by SDS-PAGE, and analyzed by immunoblotting. To control for an equal loading of cell lysate, protein concentration was determined by BCA assay (Pierce Biotechnology) according to the manufacturer’s instruction, using BSA as a standard. SDS-PAGE resolved samples were transferred to a nitrocellulose membrane and probed with primary (anti-Bcl2 and Bcl-xL; DakoCytomation) and HRP-conjugated secondary Ab (DakoCytomation), followed by ECL reagent (ECL) detection (Amersham Biosciences).

Ig binding affinity determination by competition ELISA

Increasing amounts of CIDR1{alpha} protein (3 x 10–8 to 5 x 10–6 M) were incubated for 2 h at room temperature with a constant concentration of IgG or IgM (5 µg/ml) (Jackson ImmunoResearch Laboratories) in PBS-Tween 0.05%. The mixture was thereafter added to wells precoated with 1.2 µg of CIDR1{alpha}. Following incubation at room temperature for 1 h, alkaline phosphatase-conjugated anti-human IgM or anti-human IgG were added, and the plates were further incubated for 1 h. The concentration of Ag which gave half-maximum absorption at 405 nm was determined by linear regression and regarded as the value of the dissociation constant (Kd) that corresponds to an association constant (Ka) of 1/Kd. Values were computed graphically using four-parameter sigmoidal curve fitting.

RNA preparation and real-time PCR

Total RNA was isolated from frozen B cell pellets after 12 h of culture in medium alone or containing GST or CIDR1{alpha}. The RNA was extracted with Qiagen’s RNeasy mini-kit and reverse-transcribed using TaqMan RT reagents (Applied Biosystems) according to the manufacturer’s instructions. Real-time PCR was performed using predesigned assays (Applied Biosystems) for bcl-xL and X-linked inhibitor of apoptosis (XIAP) and a custom-designed assay for a proliferation-inducing ligand (APRIL). The GAPDH gene was used as an endogenous control. Gene-specific PCR products were measured using an ABI PRISM 7700 (Applied Biosystems) sequence detection system and analyzed with ABI PRISM 7000 SDS software. With the help of a standard curve, cycle threshold (CT) values were used to determine the corresponding mRNA quantities in each sample. Samples with a CT ≥ 35 were excluded from the analysis. Results were normalized for GADPH gene expression and therefore expressed as relative mRNA expression.

DNA microarray analysis

Microarray slides (KTH HUM 29.8k) were obtained from The Royal Institute of Technology, Sweden (<www.biotech.kth.se/molbio/microarray/index.html> and <www.ebi.ac.uk/arrayexpress/>; accession no. A-MEXP-114). Differential gene expression was investigated using two-color hybridization scheme in three independent experiments performed with B cells from different donors. B cells were cultured for 24 h in medium alone or supplemented with recombinant CIDR1{alpha}, GST, or anti-Ig, and RNA was extracted using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. Residual DNA was removed by treatment with DNA-free (Ambion). The RNA quality was checked with an Agilent 2100 Bioanalyser. RNA (~2 µg) was reverse transcribed into cDNA with Superscript III RNase H– reverse transcriptase (Invitrogen Life Technologies), sonicated for 30 s, purified using Microcon filters (YM-30; Millipore), and subsequently labeled with Cy3-dCTP or Cy5-dCTP by random priming using the Klenow fragment (40 U/ml; Invitrogen Life Technologies) at 37°C for 2.5 h. The reactions were stopped by the addition of 0.5 M EDTA (pH 8.0), and unincorporated nucleotides were removed using Microcon filters (YM-30). The two fluorescent probes were mixed with hybridization buffer, denatured at 95°C for 2 min, and vacuum-concentrated for 1 min. Hybridization and washings were performed using HS400 hybridization station (TECAN); thereafter, the slides were scanned with a GenePix 4000 A scanner (Axon Instruments); at least three hybridizations (with at least one dye-swap experiment) were performed. Expression data from replicate microarray hybridization showed a high degree of reproducibility with correlation coefficients ~0.8 (data not shown). Fluorescent spot and local background intensities were quantified using Genepix Pro 5.1 software (Axon Instruments). Spots that passed the quality controls (i.e., 55% of pixel intensities should be greater than background + 1 SD and the signal intensity <3% of the saturation level for each channel), together with visual inspection, were analyzed using GeneSpring 6.1 software (Silicon Genetics). Local background was subtracted from spot signals, and fluorescence ratios were calculated. To compensate for unequal dye incorporation or any effect of the amount of template, data normalization was done by LOWESS, an intensity-dependent normalization approach. The complete data set is available at <www.ebi.ac.uk/arrayexpress/> (accession no. E-MEXP-316). The data were selected using three different filters: 1) by flags removing genes that were absent in any replicate (no signal detected), 2) by expression level to remove those genes that were deemed to be unchanging between log values 0.5 and 2.0 (>2-fold difference), and 3) by confidence using a one-sample t test against the baseline value of 1. Hierarchical clustering of gene expression profiles was performed using the Pearson correlation. Analysis of gene expression profile was conducted on three groups corresponding to different experimental conditions: CIDR1{alpha} vs GST, CIDR1{alpha} vs medium, and anti-Ig vs medium. A first unsupervised gene expression analysis was made on all three combinations, given the similarity between CIDR1{alpha} vs GST, CIDR1{alpha} vs medium signatures, the supervised analysis was conducted only on CIDR1{alpha} vs GST and anti-Ig vs medium. For comparison between CIDR1{alpha} vs GST and anti-Ig vs medium, the genes up- or down-regulated in CIDR1{alpha} vs GST were selected and compared with the expression of the same genes in anti-Ig vs medium. Because the number of replicates of each comparison is limited, we applied the Cross-Gene Error Model (provided in the GeneSpring software), which provides a more accurate estimate for the precision of a expression data by combining measurement variation and between-sample variation information.

Semiquantitative RT-PCR

To verify the microarray results, semiquantitative RT-PCR was performed, to measure gene expression of XCL1, GPS1, CDK4, and POLE4 mRNAs on B cell cultured for 24 h. RNA extraction, DNase treatment, and reverse transcription were performed as previously described. The PCR was performed using Platinum Taq polymerase (Invitrogen Life Technologies) with oligonucleotide primer pairs specific for XCL1 (forward, 5'-GCAGAATCAAGACCTACACCATCAC-3'; reverse, 5'-ATTGCTGGGTTCCTGTTGGC-3'), GSP1 (forward, 5'-CACAGGTCCGAGACATCATCTTC-3'; reverse, 5'-CCTTGCCCATCAACAGAGACTTC-3'), CDK4 (forward, 5'-AGGCTTTTGAGCATCCCAATG-3'; reverse, 5'-CCACCACTTGTCACCAGAATGTTC-3'), POLE4 (forward, 5'-CCATCTTCATTCTGGCACG-3'; reverse, 5'-GCATTATCCAAGTCTCTCCTCTG-3'), beta-actin (forward, 5'-ACTGTGCCCATCTACGAGGGGTAT-3'; reverse: 5'-TCCTTAATGTCACGCACGATTTCC-3'). The PCR were denaturated at 95°C/3 min and temperature cycled at 95°C/30 s, 56°C/30 s and 68°C/45 s for 28–30 cycles. All data were normalized to an internal housekeeping beta-actin control, and the linear amplification range for each gene was tested on the adjusted cDNA. Quantification was done using the Quantity One Software (Bio-Rad).

Statistical analysis

A Student paired two-tailed t test was performed. A value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CIDR1{alpha} preferentially activates the memory B cell compartment

To determine the effect of CIDR1{alpha} on the composition of different B lymphocyte subsets in peripheral blood, we analyzed and compared the proportion and expression of activation markers in the memory and naive compartments 48 h following CIDR1{alpha} stimulation. The proportion of CD27+ (memory B cells) and IgD+CD27– (naive B cells) varied among different donors (data not shown). Within the memory B cell compartment, the relative proportion of cells expressing the activation markers CD70 (CD27+CD70+) and CD95 (CD27+CD95+) increased between 2 and 4%, and 8 and 10%, respectively, following CIDR1{alpha} stimulation (Fig. 1). These changes are modest, but both consistent and statistically significant (p < 0.05). In contrast, CIDR1{alpha} did not significantly affect the proportion of CD27– naive B cells, independently from their activation status (Fig. 1). However, in all experiments, CIDR1{alpha} stimulation induced a low but consistent increase in the relative proportion of B cells expressing both IgD and CD27; such cells seem to play a crucial in the secondary immune response by producing high-affinity IgM (18) (Fig. 1).


Figure 1
View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 1. CIDR1{alpha} preferentially activates memory (CD27+) B cells. CD19+ B cells were cultured for 48 h in medium alone, or containing GST or CIDR1{alpha}, and stained with FITC- or PE-conjugated anti-human Abs. A, Density plot analysis of the proportion of activated memory B cell subsets. Data show one representative of eight independent experiments. B, Histogram of the median values (±SD) of the proportion of naive (CD27–) and memory (CD27+) B cells expressing different markers (CD70, CD95, IgD). {square} and {blacksquare}, Data relative to the GST and CIDR1{alpha}-treated B cells, respectively. Statistical significance was determined by a two-tailed paired Student t test on data collected from eight independent experiments with eight donors (*, p < 0.05).

 
Analysis of the levels of expression of activation markers showed that, in the memory subpopulation (CD27+), CIDR1{alpha} enhanced the expression of the activation markers CD70, CD95, CD69, the costimulatory molecule CD86, and HLA-DR (Fig. 2A) without affecting the expression of CD23, CD27, CD54, CD80, and HLA-ABC molecules (data not shown). In the naive subpopulation (CD27–), CIDR1{alpha} increased the expression of HLA-DR and induced down-regulation of HLA-ABC expression, whereas the level of expression of IgD and the activation molecules CD95, CD70 were not significantly altered (Fig. 2B). The level of the activation molecules CD23 and CD69 and of the costimulatory molecules CD54, CD80, and CD86 were not affected (data not shown). It is not likely that the difference in responsiveness to CIDR1{alpha} obeys a different kinetic between naive and memory B cells. Naive B cells show a very modest activation at 24 h (see Fig. 2B) that with the time (48 h) disappears. Initial kinetic studies performed on total B cells did not reveal a change in the response, or a biphasic response when the cultures were analyzed for up to 5 days. Furthermore, naive and memory B cells have different thresholds of activation (19, 20).


Figure 2
View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 2. Phenotypic changes of memory and naive B lymphocytes stimulated with CIDR1{alpha}. CD19+ B cells were cultured for 48 h in medium alone, or containing GST or CIDR1{alpha}, and stained with FITC- or PE-conjugated anti-human Abs. Histograms show median values of the mean intensity fluorescence (MFI ± SD) of the markers analyzed on the following: the memory population, CD27+ (A); the naive population, CD27– (B). Statistical significance was determined by a two-tailed paired Student t test on data collected from eight independent experiments (*, p < 0.05).

 
Together, the data indicate that CIDR1{alpha} preferentially activates the memory B cell compartment.

CIDR1{alpha} increases B cell survival and drives cells into cell cycle

To analyze the effects of CIDR1{alpha} on the survival and cell cycle progression of primary B cells, tonsillar B lymphocytes were cultured in the presence of GST or CIDR1{alpha} for 24–48 h, and their DNA content was measured by PI staining. Tonsillar B cell populations contain germinal center (GC) cells that are prone to undergo spontaneous apoptosis when cultured (17). At time 0, immediately after isolation, the level of cell death was negligible. At the earliest time point analyzed, 24 h, the survival was higher in the CIDR1{alpha} cultures compared with GST and medium control cultures (Fig. 3).


Figure 3
View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 3. CIDR1{alpha} rescues tonsillary B cells from spontaneous cell death. Unstimulated B cells were cultured for 24 h in medium alone or containing GST, CIDR1{alpha}, anti-Ig, or PMA plus ionomycin. Histograms illustrate the DNA content measured by PI incorporation. The numbers represent the percentage of cell death (<G0/G1) and cells in S, G2/M phases are indicated as cycling cells. The percentage of rescue from cell death was calculated as follows: 1 – (percentage of cell death in CIDR cultures/percentage of cell death in control cultures) x 100. Data are representative of one of six independent experiments.

 
In six experiments, the median percentage ± SD of dead cells (<G0/G1) in the CIDR1{alpha} culture at 24 h was 39 ± 15%, whereas in the medium and in GST cultures, it was 50 ± 16 and 55 ± 18%, respectively (Fig. 4). The median CIDR1{alpha}-induced relative protection from cell death was 22 and 29% when compared with medium and GST, respectively (p < 0.05). Treatment with anti-Ig, known to rescue B cells from death, induced a median protection of 31%. By 48 h, the effect of CIDR1{alpha} on cell survival was no longer evident, although the rescue mediated by anti-Ig was still seen (data not shown).


Figure 4
View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 4. CIDR1{alpha} promotes cell cycle entry. Percentage of B cells in the different phases of the cell cycle after 24 h of culture in medium alone or containing the following: CIDR1{alpha}, GST, anti-Ig, or PMA plus ionomycin. Cell cycle analysis was performed after PI staining. Median values of six independent experiments are shown.

 
We conclude that CIDR1{alpha} mediates a degree of protection from apoptosis in B lymphocytes, equal to, although more transient than, that induced by anti-Ig. Along with the rescue from cell death, CIDR1{alpha} induced cell cycle progression with a modest although consistent increase in the proportion of cycling cells (S+G2/M). In the CIDR1{alpha}-treated cultures, the percentage of cycling cells was 6.6% (3 ± 2% in S phase, 3 ± 2% in G2/M phase), whereas in the GST and medium cultures the values were 4% (2 ± 2% in S phase, 2 ± 2% in G2/M phase) (Fig. 4). It could be argued that the enhanced survival seen in CIDR1{alpha} cultures reflects an increased number of viable cells due to more cell division rather than reflecting protection from death. This assumption is unlikely because the proportion of cells that have undergone cell division at 24 h is negligible.

The proportions of resting cells in G0/G1 phase were as follows: 67 ± 19% in CIDR1{alpha}-treated cells, 41 ± 15% in the GST, and 46 ± 18% in medium control cultures (Fig. 4). B cells treated with anti-Ig and PMA plus ionomycin had ~2-fold increase in the amount of cells that progressed toward the G2/M phase compared with the medium control. The differences between CIDR1{alpha} treatment and medium or GST control, regarding the proportion of cells in S, G2/M, and G0/G1 phase, were statistically significant (p < 0.05).

CIDR1{alpha} has a low binding affinity for Igs

We have previously shown that CIDR1{alpha} binds to and activates B cells, an interaction mediated, at least in part, through the binding to surface IgM and IgG (10). To further analyze the binding of CIDR1{alpha} to human Ig and obtain an estimation of the affinity of the interaction, we performed competition ELISA experiments. Fig. 5 shows results relative to the percentage of IgM and IgG binding to CIDR1{alpha}. The affinity values obtained by calculation in competing conditions were Ka {approx} 3.7 x 106 M–1 (Kd = 2.68 ± 0.30 x 10–7 M) for IgM and Ka {approx} 1.2 x 106 M–1 (Kd = 8.57 ± 0.89 x 10–7 M) for IgG. These Ka values place the CIDR1{alpha} among the low-affinity Ags, although able to allow BCR triggering (21).


Figure 5
View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 5. Evaluation of CIDR1{alpha} affinity for human IgG and IgM by competition ELISA. Inhibition of human Ig binding (IgG and IgM) to immobilized CIDR1{alpha} was evaluated in a competition ELISA as described in Materials and Methods. Values were computed graphically using four-parameter sigmoidal curve fitting. Curves shown are representative of six independent experiments.

 
The impact of CIDR1{alpha} on genes that regulate the apoptotic pathway

Given the capacity of CIDR1{alpha} to protect B cells from cell death, we investigated the effect of CIDR1{alpha} on the expression of some genes that regulate cell survival and apoptosis such as bcl2, bcl-xL, APRIL, and XIAP (22). We quantified and compared by quantitative real-time PCR the mRNA expression levels of bcl-xL, APRIL, and XIAP. CIDR1{alpha} treatment did not affect bcl-xL mRNA levels and produced a modest decrease of APRIL mRNA, significantly increasing XIAP mRNA (p < 0.05) (Fig. 6A). At the protein level, CIDR1{alpha} produced down-modulation of Bcl-xL with a slight down-regulation of Bcl2 expression (Fig. 6B). Thus, the protection from cell death induced by CIDR1{alpha} may be mediated by inducing the expression of the antiapoptotic regulator XIAP without affecting the bcl2, bcl-xL intrinsic antiapoptotic pathway. This protective effect appears to be different from the one mediated by the anti-Ig.


Figure 6
View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 6. Effect of CIDR1{alpha} on Bcl2, Bcl-xL, APRIL, and XIAP expression. B cells were cultured for 12–24 h in medium alone or containing GST, CIDR1{alpha}, or anti-Ig. A, Expression of the mRNA levels of bcl-xL, APRIL, and XIAP was performed by quantitative real-time PCR after 12 h of culture. Data are expressed as relative to the expression of GAPDH mRNA. B, Expression of the antiapoptotic proteins Bcl2 and Bcl-xL was evaluated by Western blot after 24 h of culture. Statistical significance was determined by a two-tailed paired Student t test (*, p < 0.05).

 
CIDR1{alpha} and Ig activation generate a different gene expression profile

In our previous studies, competition experiments demonstrated that the CIDR1{alpha} induced B cell activation is only partially mediated by its binding to Igs (10). Thus, it became of interest to analyze and compare the gene expression profiles induced by CIDR1{alpha} and anti-Ig. To this end, three comparative analyses were performed: CIDR1{alpha} vs GST, CIDR1{alpha} vs medium, and anti-Ig vs medium. First, we conducted an unsupervised analysis of the CIDR1{alpha} vs GST profile, compared with the CIDR1{alpha} vs medium and identified a high degree of homology (95%) (data not shown). This degree of homology between the two profiles confirmed that, in our experimental conditions, the effect of GST on B cell responses is minor; hence, we will only refer to the comparisons between CIDR1{alpha} vs GST and anti-Ig vs medium. To compare the gene expression signatures induced by CIDR1{alpha} and anti-Ig, we performed a supervised analysis and selected those genes up- or down-regulated by CIDR1{alpha} (CIDR1{alpha} vs GST). The same set of genes was then analyzed in anti-Ig vs medium, and the two expression profiles were compared (Fig. 7A).


Figure 7
View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 7. Gene expression profile comparison between CIDR1{alpha} and Ig stimulation on B lymphocytes. Cluster analysis of gene expression profile of B cells in two different experimental conditions: CIDR1{alpha} vs GST (CG) and anti-Ig vs medium (IM) (see Materials and Methods). A, Tree view display of the results of 377 differentially expressed genes under CG condition (with the positive expression values appearing in all two conditions) as described in Materials and Methods. Each row represents a gene; each column represents a sample. The level of expression of each gene in each sample is represented using a red-blue color scale (red, high expression). B, Enlargement of the Tree view display of the genes showing up-regulation (>2-fold change) in both conditions. The data were processed with the GeneSpring 6.1 software. The color scale is based on the signal ratio in between each condition, and ranges between 0 (blue) and 6 (red), where red indicates up-regulation and blue indicates down-regulation. C, Validation of the microarray results by semiquantitative PCR. Transcription level of two up-regulated genes (XCL1 and GPS1) and two down-regulated genes (CDK4 and POLE4) were verified by semiquantitative PCR, to validate the microarray analysis (left panel). The expected sizes of XCL1, GPS1, CDK4, POLE4, and beta-actin RT-PCR products were 186, 363, 265, 118, and 158 bp, respectively. The histograms in the right panel show the relative gene expression levels after normalization against the housekeeping beta-actin gene (bottom). Expressions relative to CIDR{alpha}-treated B cells are shown in dark gray (the numbers above the significance bar show the fold increase/decrease in CIDR1{alpha} vs GST, and the asterisk (*) indicates the consistency of CIDR1{alpha} vs GST compared with the array data).

 
The immunological signatures of CIDR1{alpha} and anti-Ig showed a high number of differentially expressed genes. Although the majority (97%) of the genes listed in Fig. 7A were differentially expressed in the two groups (CIDR1{alpha} vs GST and anti-Ig vs medium), only a small number of genes (n = 8) were common. Interestingly, these eight genes were all up-regulated (Fig. 7B, Table I). Among them, U2AF2, ZNF135, and SLC39A3 indicate activation-induced gene transcription. GSTM2 is commonly associated with inhibition of cell death and activation of different signaling pathways, such as the MEK pathway. The other four genes do not as yet appear to have known function or apparent relation to the activation process.


View this table:
[in this window]
[in a new window]
  		Table I. Genes up-regulated by both CIDR1{alpha} and anti-Ig

 
CIDR1{alpha}-treated B cells display an activated gene expression profile

To further understand the cellular processes altered by CIDR1{alpha}, we analyzed and compared the gene expression profile of CIDR1{alpha} and GST control-treated B cells. We examined the expression of ~29,800 genes using the human microarray KTH HUM 29.8k. Analysis of the data established that a total of 377 genes exhibited different and significantly altered gene expression between CIDR1{alpha} and GST cultured B cells. These included 105 known genes, of which 81 were up-regulated (Table II) and 24 down-regulated (Table III). A cluster analysis of known genes made according to their cellular function (determined by the gene information: SwissProtID (ExpASy Proteomics Server; <www.expasy.org>) and PubMed search), allowed classification into five groups: cell growth/apoptosis; immune response; transcription and translation; immunological signaling; and cell communication (Tables II and III). The largest groups were represented by genes involved in immunological signaling pathways, cell growth and apoptosis, and transcription.


View this table:
[in this window]
[in a new window]
  		Table II. Genes up-regulated by CIDR1{alpha} (CIDR vs GST)

 

View this table:
[in this window]
[in a new window]
  		Table III. Genes down-regulated by CIDR1{alpha} (CIDR vs GST)

 
Up-regulated genes induced by CIDR1{alpha} includes several genes previously described as mitogen-activated, involved in pathways that control cell growth/apoptosis, transcription/translation, and that are normally induced during immune responses. The up-regulation of both TRAF3 and TRAF4 suggests activation of the NF-{kappa}B pathway. The increased expression of two of the most important protein kinases, PKC and the PKA related forms (PRKAG3, PRKX), is in line with the observed CIDR1{alpha}-mediated up-regulation of genes involved in different signaling pathways; among them: the MEK/ERK pathway (EIF2B5), the MEKK/JNK pathway (TRAF3, GPS1), and the MKK/MAPK pathways (MAPK4K5, IQGAP1, IQGAP2). These signaling pathways lead to activation and survival are involved in B cell immune responses. CIDR1{alpha} may inhibit the JAK/STAT pathway as indicated by an increased expression in the negative regulators PIAS2 and SOCS1. The coordinated regulation of these different pathways results in B cell activation as reflected by the activated phenotype (Fig. 2).

CIDR1{alpha} stimulation also increased the gene expression of numerous transcription factors including DOTL-1, Ets-2 (ETV1), TLE4, and many members of the ZNF family (ZNF36, ZNF135, ZNF318, ZNF263) (Table II). The concerted action of these transcription factors is crucial for the processes that lead to differentiation of mature B cells into Ig-secreting plasma cells and memory B cells (23, 24, 25). Up-regulated genes strictly correlated to immune responses included: chemokine and chemokine receptors such as the lymphoactin XCL1 and CCRL2, often expressed during B cell activation and migration to secondary lymph nodes; the IL-7R (IL7R) expressed during different phases of B cell development; the insulin-like growth factor binding protein 5 (IGFBP5); and the IL-18 expressed on naive, memory, and GC B cells upon activation. The up-regulation of the HLA-DQ is indicative of B cell activation. Other up-regulated genes are involved in early and late events of cell growth and apoptosis. CALB1 or calbindin is an important Ca2+ buffer molecule and early mediator of cellular activation known to inhibit numerous apoptotic stimuli. Later mediators are DAPK2, DNAJC7 also known as Hsp40, and HRK or hara-kiri whose overexpression can either induce or suppress apoptosis. FBXO6 and FPGS genes are involved in the cell cycle control and proliferation.

A small number of genes with known function were down-regulated by CIDR1{alpha}; few of them have been described in association with B lymphocyte functions. Genes functionally clustered in the cell growth/apoptosis were CDK4, important for cell cycle progression; RP9, described in relation to B cell proliferation; and SAV1 and TNFSF8 (CD30). SAV1 promotes exit from cell cycle and together with TNFSF8 induces apoptosis. Two genes were classified as part of the immune response group, CD36 and HLA-A: the latter for which we had noted down-regulation of surface protein expression (Fig. 2B). To confirm the array data, we run a semiquantitative RT-PCR on two up-regulated (XCL1, GPS1) and two down-regulated (CDK4, POLE4) genes. The data showed an increase/decrease of gene expression consistent with the one seen in the array (Fig. 7C). Taken together, the microarray data are consistent with the phenotypic and functional analysis demonstrating that CIDR1{alpha} activates human B lymphocytes. However, the signaling pathways induced by CIDR1{alpha} do not seem to be the same as those induced by BCR (anti-Ig signaling) as reflected by the different gene expression profiles.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Although the impact of P. falciparum malaria on B cell-mediated immunity has been recognized for ~40 years (1), little is known about the identity of the Ags and the mechanisms that underlie the production of large amounts of nonspecific Igs during P. falciparum malaria infection. Our recent identification of the CIDR1{alpha} domain of PfEMP1 as a polyclonal B cell activator and as an IBP led us to investigate further its effects on the B cell compartment. The data presented in this paper extend our previous results and, importantly, demonstrate that CIDR1{alpha} preferentially activates memory B cells and protects B cells from apoptosis.

It could be argued that because CIDR1{alpha} fusion protein was produced in E. coli, the activation observed could be due to contaminating endotoxin. We can rule out this possibility because the proliferative response to the recombinant Ag preparations was not affected by the presence of polymyxin B or LPS. Furthermore, human B cells, in contrast to murine B cells, do not respond to endotoxin or LPS, because they do not express the TLR-2 and -4, essential components of the LPS receptor signaling complex (26).

The preferential activation of the memory compartment may relate to their lower threshold for activation, and is in line with the results of Bernasconi et al. (19), who demonstrated that memory B lymphocytes selectively proliferate and differentiate into plasma cells in vitro in response to polyclonal stimuli in the absence of BCR triggering; whereas naive B cells require specific BCR triggering. These results led to the hypothesis that one of the mechanisms for the maintenance of serological memory involves continuous activation of memory B cells by polyclonal B cell activators (19). Most likely, CIDR1{alpha}, as a polyclonal B cell activator, contributes in a similar fashion to the hyperglobulinemia that characterizes chronic malaria infection (1). It should be noted that, in vivo, the consequence of CIDR1{alpha} interacting with B lymphocytes may be potentiated by costimulation and bystander T cell help (19). During a malaria episode, IE are trapped in the spleen where splenic B cells may be activated by CIDR1{alpha} expressed on the IE surface, and presented as soluble Ag, or as immune complexes, by dendritic cells in the presence of cytokines, T cell help, and costimulatory signals. In contrast, the polyclonal memory B cell activation induced by CIDR1{alpha} may impair the maintenance of Ag-specific memory B cells. Thus, the suggested role of microbial IBPs, and thereby of CIDR1{alpha}, as an evasion mechanism to divert specific Ab responses (14, 15) is in line with the lack of specific Ig memory observed in children from malaria endemic areas (27).

CIDR1{alpha} protects a significant proportion of B cells from spontaneous cell death, an outcome that is accompanied by the up-regulation of the antiapoptotic factor XIAP. The contention that the two observations are linked is supported by the up-regulation of numerous genes involved in the activation of the ERK/MAPK pathway (Table II), known to be associated with suppression of proapoptotic signaling and sustaining XIAP levels (28). Following CIDR1{alpha} activation, a large proportion of B cells stay in the G0/G1 phase with concomitant protection from spontaneous apoptosis. However, the given signal is sufficient to allow a small proportion of cells to proceed beyond the S phase restriction point leading to proliferation. Polyclonal activators stimulate rapid G1 entry, protection from apoptosis, and S phase entry, which is dependent on B cell differentiation state and the Ag affinity (29, 30, 31). A signal generated through surface Ig can lead to proliferation or apoptosis, depending on the maturation stage of the responding B cell, and on additional signals provided by costimulation and T cell help. In vivo, the antiapoptotic and B cell activation properties of CIDR1{alpha} may be potentiated by other not-yet-identified polyclonal B cell activators present in P. falciparum-infected cells or by additional B cell stimulatory factors such as IL-10, IgE, known to be produced during malaria infection (32). The occurrence of protracted malaria episodes is an additive factor that may enhance and perpetuate the B cell stimulation that finally leads to malignant development.

CIDR1{alpha} effect, as polyclonal B cell activator, is partially mediated through binding to surface Ig (10). In vitro, CIDR1{alpha} also binds to CD36, and PECAM-1/CD31 (6, 8, 16); thus, the effect of CIDR1{alpha} on the cell cycle may involve not only the interaction with surface Ig but also the binding to other receptors that affect the decision to proceed to activation.

The binding affinity of an Ag is important for the outcome of activation. The binding affinity of CIDR1{alpha} to IgG and IgM classifies it as a low-affinity Ag. However, the same level of affinity, has been demonstrated to be sufficient to drive B cell proliferation and differentiation during T-independent immune responses (21). Indeed, Ags with very low affinity for the BCR (Ka {approx} 105 M–1) can transduce activation signals (33, 34, 35). During T-independent immune responses, large differences in affinity (Ka {approx} 105–106 M–1) produce only small variations in the intrinsic ability of B cells to mount a productive response to Ag (21). The in vivo impact of an Ag results from a combination of its affinity and persistence. A low antigenic clearance of CIDR1{alpha}, due to high malaria endemicity, could result in a long persistence and therefore augment its impact on B cell responses.

Despite its ability to bind Igs, characterization and comparison of the gene expression profile induced by both CIDR1{alpha} and anti-Ig activation demonstrated that there was a large difference in the signatures imposed by both stimuli. These results indicate that either the signaling by CIDR1{alpha} proceeds via receptor(s) other than Ig, or concomitantly through Ig with additional receptors. Alternatively, the different affinities of CIDR1{alpha} and anti-Ig, or possibly epitopes engaged, may lead to different signaling profiles.

In conclusion, the combination of gene expression profiling with phenotypic data and cell cycle analysis has enabled us to gain a better understanding of the mechanisms that drive malaria polyclonal B cell activation. The observed activation of memory B cells mediated by CIDR1{alpha}, has to be considered for a rational design of a malaria vaccine, because it could lead to inappropriate Ab production and hyperglobulinemia. The polyclonal B cell activation and augmented survival induced by CIDR1{alpha} is of relevance for understanding the mechanisms behind the increased risk of Burkitt’s lymphoma in malaria endemic areas (36). A possible role of malaria infection in increasing the risk of endemic Burkitt’s lymphoma may relate to its capacity to augment the survival of GC B cells that carry translocations.


   Acknowledgments
 
We thank Drs. Alf Grandien and Lyda M. Osorio for interesting discussions and critical review of this manuscript. We also thank Mia Löwbeer for expert technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Swedish International Development Cooperation Agency, Barncancerfonden, the Swedish Research Council, the Swedish Foundation for Strategic Research, and the Karolinska Institutet. Back

2 Address correspondence and reprint requests to Dr. Daria Donati, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, F59, SE-141 86 Stockholm, Sweden. E-mail address: daria.donati{at}ki.se Back

3 Abbreviations used in this paper: IE, infected erythrocyte; PfEMP1, Plasmodium falciparum erythrocyte membrane protein 1; IBP, Ig binding protein; CIDR1{alpha}, cysteine-rich interdomain region 1{alpha}; PI, propidium iodide; XIAP, X-linked inhibitor of apoptosis; APRIL, a proliferation-inducing ligand; GC, germinal center; CT, cycle threshold. Back

Received for publication May 24, 2005. Accepted for publication May 25, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Abele, D. C., J. E. Tobie, G. J. Hill, P. G. Contacos, C. B. Evans. 1965. Alterations in serum proteins and 19S antibody production during the course of induced malaria infections in man. Am. J. Trop. Med. Hyg. 14: 191-197. [Abstract/Free Full Text]
  2. Adu, D., D. G. Williams, I. A. Quakyi, A. Voller, Y. Anim-Addo, A. A. Bruce-Tagoe, G. D. Johnson, E. J. Holborow. 1982. Anti-ssDNA and antinuclear antibodies in human malaria. Clin. Exp. Immunol. 49: 310-316. [Medline]
  3. McGregor, I. A., H. M. Giles, J. H. Walter, A. H. Davies. 1956. Effects of heavy and repeated malaria infections of Gambian infants and children. Br. Med. J. 2: 686-691.
  4. Greenwood, B. M., R. M. Vick. 1975. Evidence for a malaria mitogen in human malaria. Nature 257: 592-594. [Medline]
  5. Bates, I., G. Bedu-Addo. 1997. Chronic malaria and splenic lymphoma: clues to understanding lymphoma evolution. Leukemia 11: 2162-2167. [Medline]
  6. Scholander, C., J. Carlson, P. G. Kremsner, M. Wahlgren. 1998. Extensive immunoglobulin binding of Plasmodium falciparum-infected erythrocytes in a group of children with moderate anemia. Infect. Immun. 66: 361-363. [Abstract/Free Full Text]
  7. MacLennan, I. C.. 1994. Germinal centers. Annu. Rev. Immunol. 12: 117-139. [Medline]
  8. Chen, Q., A. Heddini, A. Barragan, V. Fernandez, S. F. Pearce, M. Wahlgren. 2000. The semiconserved head structure of Plasmodium falciparum erythrocyte membrane protein 1 mediates binding to multiple independent host receptors. J. Exp. Med. 192: 1-10. [Abstract/Free Full Text]
  9. Flick, K., C. Scholander, Q. Chen, V. Fernandez, B. Pouvelle, J. Gysin, M. Wahlgren. 2001. Role of nonimmune IgG bound to PfEMP1 in placental malaria. Science 293: 2098-2100. [Abstract/Free Full Text]
  10. Donati, D., L. P. Zhang, Q. Chen, A. Chene, K. Flick, M. Nystrom, M. Wahlgren, M. T. Bejarano. 2004. Identification of a polyclonal B-cell activator in Plasmodium falciparum. Infect. Immun. 72: 5412-5418. [Abstract/Free Full Text]
  11. Romagnani, S., M. G. Giudizi, G. del Prete, E. Maggi, R. Biagiotti, F. Almerigogna, M. Ricci. 1982. Demonstration on protein A of two distinct immunoglobulin-binding sites and their role in the mitogenic activity of Staphylococcus aureus Cowan I on human B cells. J. Immunol. 129: 596-602. [Medline]
  12. Widders, P. R.. 1990. Fc Receptors and the Pathogenesis of Bacterial Infections in Animals Academic, Orlando, FL.
  13. Langone, J. J.. 1982. Protein A of Staphylococcus aureus and related immunoglobulin receptors produced by streptococci and pneumonococci. Adv. Immunol. 32: 157-252. [Medline]
  14. Leonetti, M., J. Galon, R. Thai, C. Sautes-Fridman, G. Moine, A. Menez. 1999. Presentation of antigen in immune complexes is boosted by soluble bacterial immunoglobulin binding proteins. J. Exp. Med. 189: 1217-1228. [Abstract/Free Full Text]
  15. Daniel-Ribeiro, C., J. de Oliveira-Ferreira, D. M. Banic, B. Galvao-Castro. 1989. Can malaria-associated polyclonal B-lymphocyte activation interfere with the development of anti-sporozoite specific immunity?. Trans. R. Soc. Trop. Med. Hyg. 83: 289-292. [Medline]
  16. Chen, Q., A. Barragan, V. Fernandez, A. Sundstrom, M. Schlichtherle, A. Sahlen, J. Carlson, S. Datta, M. Wahlgren. 1998. Identification of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) as the rosetting ligand of the malaria parasite P. falciparum. J. Exp. Med. 187: 15-23. [Abstract/Free Full Text]
  17. Pound, J. D., J. Gordon. 1997. Maintenance of human germinal center B cells in vitro. Blood 89: 919-928. [Abstract/Free Full Text]
  18. Shi, Y., K. Agematsu, H. D. Ochs, K. Sugane. 2003. Functional analysis of human memory B-cell subpopulations: IgD+CD27+ B cells are crucial in secondary immune response by producing high affinity IgM. Clin. Immunol. 108: 128-137. [Medline]
  19. Bernasconi, N. L., E. Traggiai, A. Lanzavecchia. 2002. Maintenance of serological memory by polyclonal activation of human memory B cells. Science 298: 2199-2202. [Abstract/Free Full Text]
  20. Tangye, S. G., D. T. Avery, E. K. Deenick, P. D. Hodgkin. 2003. Intrinsic differences in the proliferation of naive and memory human B cells as a mechanism for enhanced secondary immune responses. J. Immunol. 170: 686-694. [Abstract/Free Full Text]
  21. Shih, T. A., M. Roederer, M. C. Nussenzweig. 2002. Role of antigen receptor affinity in T cell-independent antibody responses in vivo. Nat. Immunol. 3: 399-406. [Medline]
  22. Rathmell, J. C.. 2004. B-cell homeostasis: digital survival or analog growth?. Immunol. Rev. 197: 116-128. [Medline]
  23. Glimcher, L. H., H. Singh. 1999. Transcription factors in lymphocyte development—T and B cells get together. Cell 96: 13-23. [Medline]
  24. Clevers, H. C., R. Grosschedl. 1996. Transcriptional control of lymphoid development: lessons from gene targeting. Immunol. Today 17: 336-343. [Medline]
  25. Fitzsimmons, D., J. Hagman. 1996. Regulation of gene expression at early stages of B-cell and T-cell differentiation. Curr. Opin. Immunol. 8: 166-174. [Medline]
  26. Muzio, M., D. Bosisio, N. Polentarutti, G. D’Amico, A. Stoppacciaro, R. Mancinelli, C. van’t Veer, G. Penton-Rol, L. P. Ruco, P. Allavena, A. Mantovani. 2000. Differential expression and regulation of Toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. J. Immunol. 164: 5998-6004. [Abstract/Free Full Text]
  27. Dorfman, J. R., P. Bejon, F. M. Ndungu, J. Langhorne, M. M. Kortok, B. S. Lowe, T. W. Mwangi, T. N. Williams, K. Marsh. 2005. B cell memory to 3 Plasmodium falciparum blood-stage antigens in a malaria-endemic area. J. Infect. Dis. 191: 1623-1630. [Medline]
  28. Hu, P., Z. Han, A. D. Couvillon, J. H. Exton. 2004. Critical role of endogenous Akt/IAPs and MEK1/ERK pathways in counteracting endoplasmic reticulum stress-induced cell death. J. Biol. Chem. 279: 49420-49429. [Abstract/Free Full Text]
  29. Green, D. R., D. W. Scott. 1994. Activation-induced apoptosis in lymphocytes. Curr. Opin. Immunol. 6: 476-487. [Medline]
  30. Yi, A. K., M. Chang, D. W. Peckham, A. M. Krieg, R. F. Ashman. 1998. CpG oligodeoxyribonucleotides rescue mature spleen B cells from spontaneous apoptosis and promote cell cycle entry. J. Immunol. 160: 5898-5906. [Abstract/Free Full Text]
  31. Piatelli, M. J., D. Tanguay, T. L. Rothstein, T. C. Chiles. 2003. Cell cycle control mechanisms in B-1 and B-2 lymphoid subsets. Immunol. Res. 27: 31-52. [Medline]
  32. Wahlgren, M., J. S. Abrams, V. Fernandez, M. T. Bejarano, M. Azuma, M. Torii, M. Aikawa, R. J. Howard. 1995. Adhesion of Plasmodium falciparum-infected erythrocytes to human cells and secretion of cytokines (IL-1-beta, IL-1RA, IL-6, IL-8, IL-10, TGFbeta, TNF{alpha}, G-CSF, GM-CSF). Scand. J. Immunol. 42: 626-636. [Medline]
  33. Bell, G. I.. 1974. Model for the binding of multivalent antigen to cells. Nature 248: 430-431. [Medline]
  34. Armitage, R. J., M. R. Alderson. 1995. B-cell stimulation. Curr. Opin. Immunol. 7: 243-247. [Medline]
  35. George, J., S. J. Penner, J. Weber, J. Berry, J. L. Claflin. 1993. Influence of membrane Ig receptor density and affinity on B cell signaling by antigen: implications for affinity maturation. J. Immunol. 151: 5955-5965. [Abstract]
  36. Greenwood, B. M., J. H. Playfair, G. Torrigiani. 1970. Burkitt lymphoma and malaria. Lancet 2: 418-422. [Medline]



This article has been cited by other articles:


Home page
 Postgrad. Med. J.Home page
G Brady, G J MacArthur, and P J Farrell
Epstein-Barr virus and Burkitt lymphoma
Postgrad. Med. J., July 1, 2008; 84(993): 372 - 377.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Pathol.Home page
G Brady, G J MacArthur, and P J Farrell
Epstein Barr virus and Burkitt lymphoma
J. Clin. Pathol., December 1, 2007; 60(12): 1397 - 1402.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
C. L. Montes, E. V. Acosta-Rodriguez, M. C. Merino, D. A. Bermejo, and A. Gruppi
Polyclonal B cell activation in infections: infectious agents' devilry or defense mechanism of the host?
J. Leukoc. Biol., November 1, 2007; 82(5): 1027 - 1032.
[Abstract] [Full Text] [PDF]

Home page
 ASH Education BookHome page
G. L. Kelly and A. B. Rickinson
Burkitt Lymphoma: Revisiting the Pathogenesis of a Virus-Associated Malignancy
Hematology, January 1, 2007; 2007(1): 277 - 284.
[Abstract] [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 Donati, D.
Right arrow Articles by Bejarano, M. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Donati, D.
Right arrow Articles by Bejarano, M. T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS