We tested the effect of transforming growth factor (TGF)-beta 1 and TGF-beta 2 on the proliferati... more We tested the effect of transforming growth factor (TGF)-beta 1 and TGF-beta 2 on the proliferation of human B cell lines. The panel was selected to give information whether (1) their origin, (2) their phenotype, (3) their Epstein-Barr virus (EBV) carrier state, influence their responsiveness. The growth of lymphoblastoid cell lines (LCL) was not inhibited by TGF-beta 1. The EBV-carrying Burkitt lymphoma (BL) lines, Daudi, Jijoye, Rael but not Raji were inhibited. Three EBV-negative BL lines and the majority of their converted sublines were sensitive. The cell lines tested expressed TGF-beta receptors and TGF-beta 1 transcripts. The proliferation of EBV-infected B cells was inhibited by TGF-beta, their sensitivity decreased, however, after 3 days. The results suggest that the activation state of the B cells is decisive for TGF-beta sensitivity and EBV influences it indirectly by changing the cell phenotype.
Although malaria and Epstein-Barr (EBV) infection are recognized cofactors in the genesis of ende... more Although malaria and Epstein-Barr (EBV) infection are recognized cofactors in the genesis of endemic Burkitt lymphoma (BL), their relative contribution is not understood. BL, the most common paediatric cancer in equatorial Africa, is a high-grade B cell lymphoma characterized by c-myc translocation. EBV is a ubiquitous B lymphotropic virus that persists in a latent state after primary infection, and in Africa, most children have sero-converted by 3 y of age. Malaria infection profoundly affects the B cell compartment, inducing polyclonal activation and hyper-gammaglobulinemia. We recently identified the cystein-rich inter-domain region 1a (CIDR1a) of the Plasmodium falciparum membrane protein 1 as a polyclonal B cell activator that preferentially activates the memory compartment, where EBV is known to persist. Here, we have addressed the mechanisms of interaction between CIDR1a and EBV in the context of B cells. We show that CIDR1a binds to the EBV-positive B cell line Akata and increases the number of cells switching to the viral lytic cycle as measured by green fluorescent protein (GFP) expression driven by a lytic promoter. The virus production in CIDR1a-exposed cultures was directly proportional to the number of GFP-positive Akata cells (lytic EBV) and to the increased expression of the EBV lytic promoter BZLF1. Furthermore, CIDR1a stimulated the production of EBV in peripheral blood mononuclear cells derived from healthy donors and children with BL. Our results suggest that P. falciparum antigens such as CIDR1a can directly induce EBV reactivation during malaria infection that may increase the risk of BL development for children living in malaria-endemic areas. To our knowledge, this is the first report to show that a microbial protein can drive a latently infected B cell into EBV replication. Citation: Chêne A, Donati D, Guerreiro-Cacais AO, Levitsky V, Chen Q, et al. (2007) A molecular link between malaria and Epstein-Barr virus reactivation. PLoS Pathog 3(6): e80.
We have analyzed the EBV-related immune parameters of a healthy EBV-seropositive individual (ST) ... more We have analyzed the EBV-related immune parameters of a healthy EBV-seropositive individual (ST) who has regular antibody titers but defective inhibitory capacity toward the growth of autologous EBV-infected B cells. This in vitro function reflects the EBV-specific memory because it does not occur in experiments performed with cells of seronegative individuals. An analysis of events following in vitro EBV infection showed that lymphocytes of ST behaved in some tests in the same way as those collected from seronegative individuals. These parameters were: (I) lack of-γ-IFN production 24 hr after EBV infection; (2) low production of soluble factors that inhibit EBV-induced B-cell proliferation; (3) lack of generation of LCL selective cytotoxicity after repeated stimulation with autologous LCL; and (4) high proportion of EBNA-positive cells in 7-day-old EBV-infected cultures. On the other hand, cellular memory to the virus detected by the production of IL–2 24 hr after infection, and by the production of LIF upon exposure to EBV-encoded antigens, conformed with the results obtained with seropositive individuals. T-cell-mediated inhibition of EBV-induced B-cell growth in vitro has been regarded as a corollary of in vivo control of EBV-infected B cells. However, it is absent or has a low efficiency in certain disease categories which are not accompanied by risk of B-EBV growth. Our results with a healthy individual also indicate that several mechanisms contribute to a harmless lifelong virus carrier state.
Background The expression of recombinant proteins in Escherichia coli is an important and frequen... more Background The expression of recombinant proteins in Escherichia coli is an important and frequently used tool within malaria research, however, this method remains problematic. High A/T versus C/G content and frequent lysine and arginine repeats in the Plasmodium falciparum genome are thought to be the main reason for early termination in the mRNA translation process. Therefore, the majority of P. falciparum derived recombinant proteins is expressed only as truncated forms or appears as insoluble inclusion bodies within the bacterial cells. Methods Several domains of PfEMP1 genes obtained from different P. falciparum strains were expressed in E. coli as GST-fusion proteins. Expression was carried out under various culture conditions with a main focus on the time point of induction in relation to the bacterial growth stage. Results and conclusions When expressed in E. coli recombinant proteins derived from P. falciparum sequences are often truncated and tend to aggregate what in turn leads to the formation of insoluble inclusion bodies. The analysis of various factors influencing the expression revealed that the time point of induction plays a key role in successful expression of A/T rich sequences into their native conformation. Contrary to recommended procedures, initiation of expression at post-log instead of mid-log growth phase generated significantly increased amounts of soluble protein of a high quality. Furthermore, these proteins were shown to be functionally active. Other factors such as temperature, pH, bacterial proteases or the codon optimization for E. coli had little or no effect on the quality of the recombinant protein, nevertheless, optimizing these factors might be beneficial for each individual construct. In conclusion, changing the timepoint of induction and conducting expression at the post-log stage where the bacteria have entered a decelerated growth phase, greatly facilitates and improves the expression of sequences containing rare codons.
... 7.Fernandez, V., Hommel, M., Chen, Q., Hagblom, P. & Wahlgren, M. J. Exp ... Top of page.... more ... 7.Fernandez, V., Hommel, M., Chen, Q., Hagblom, P. & Wahlgren, M. J. Exp ... Top of page. Mats Wahlgren and Maria Teresa Bejarano are at the Microbiology and Tumour Biology Centre, Karolinska Institutet, and the Swedish Institute for Infectious Disease Control, Box 280, S-171 ...
Polyclonal B-cell activation and hypergammaglobulinemia are prominent features of human malaria. ... more Polyclonal B-cell activation and hypergammaglobulinemia are prominent features of human malaria. We report here that Plasmodium falciparum-infected erythrocytes directly adhere to and activate peripheral blood B cells from nonimmune donors. The infected erythrocytes employ the cysteine-rich interdomain region 1␣ (CIDR1␣) of P. falciparum erythrocyte membrane protein 1 (PfEMP1) to interact with the B cells. Stimulation with recombinant CIDR1␣ induces proliferation, an increase in B-cell size, expression of activation molecules, and secretion of immunoglobulins (immunoglobulin M) and cytokines (tumor necrosis factor alpha and interleukin-6). Furthermore, CIDR1␣ binds to Fab and Fc fragments of human immunoglobulins and to immunoglobulins purified from the sera of different animal species. This binding pattern is similar to that of the polyclonal B-cell activator Staphylococcus aureus protein A. Our findings shed light on the understanding of the molecular basis of polyclonal B-cell activation during malaria infections. The results suggest that the var gene family encoding PfEMP1 has evolved not only to mediate the sequestration of infected erythrocytes but also to manipulate the immune system to enhance the survival of the parasite.
Epstein-Barr-virus (EBV)-positive Burkitt's-lymphoma (BL) cell lines are not recognized by EBV-sp... more Epstein-Barr-virus (EBV)-positive Burkitt's-lymphoma (BL) cell lines are not recognized by EBV-specific T cells, due to their non-immunogenic phenotype and restricted expression of latent EBV genes. We tested whether triggering of CD40 can alter the phenotype of the tumor cells with regard to: (i) expression of surface markers, (ii) expression of viral antigens, (iii) presentation of endogenous antigens to MHCclass-I restricted cytotoxic T lymphocytes (CTLs), (iv) stimulatory capacity in allogeneic mixed-lymphocyte cultures (MLCs), (v) sensitivity to natural-killer (NK)-cell-mediated lysis. Co-culture of EBV-positive BL cells with CD40-ligandtransfected L cells induced up-regulation of CD54 and CD80 but did not affect the expression of viral genes. In spite of significant up-regulation of TAP1 and TAP2, and increased expression of MHC class I, the BL cells remained unable to present endogenously expressed viral antigens to EBVspecific CTL. However, the up-regulation of adhesion and co-stimulatory molecules was associated with increased stimulatory capacity in MLC and enhanced sensitivity to NK cells. These findings indicate that, while inducing only a modest phenotype shift, cross-linking of CD40 under physiologic conditions may selectively enhance the sensitivity of BL cells to anti-tumor immune responses. Int. J. Cancer 83:772-779, 1999.
Blood lymphocyte subsets of various densities from 14 healthy donors and 25 patients with solid t... more Blood lymphocyte subsets of various densities from 14 healthy donors and 25 patients with solid tumors were used in tests for cytotoxicity against allogeneic or autologous tumor cells. The allogeneic combinations comprised 36 tests on 28 tumors. In these the unmanipulated total lymphocyte population was cytotoxic in one experiment. In tests with density-separated subsets, 16 experiments were positive. The lytic effect resided in the light subsets. Auto-tumor lysis was detected in 7 of 25 cases, when the total lymphocyte population was used. Eleven additional tests showed cytotoxocity with the separated subsets. The autologous tumor cells were damaged by the light lymphocytes of 15 patients. Of these patients, 6 had cytotoxic cells also in the high-density subset. In seven cases auto-tumor lysis was exerted only by the dense lymphocytes. Thus, in 13 cases the profile of auto-tumor lysis differed from that observed with the allogeneic combinations and against K562. Pretreatment of the effectors with Hu-IFN-alpha enhanced or induced cytotoxicity in the light lymphocytes, against both allogeneic and autologous tumors, while the dense subset was rarely potentiated. The autologous system may represent an immune situation and therefore it is not unexpected that the active cells should have different phenotypic characteristics from those of the operationally natural killer system.
We have analyzed the effect of Cyclosporin-A (CsA) on the in vitro suppression of Epstein-Barr vi... more We have analyzed the effect of Cyclosporin-A (CsA) on the in vitro suppression of Epstein-Barr virus (EBV)-induced B-cell proliferation exerted by autologous T-lymphocyte subpopulations separated on the basis of cell density. CsA abolished the growth-inhibitory capacity of high-density T cells but influenced only marginally the activity of low-density lymphocytes; this suggested that different mechanisms mediate suppression in the two subsets. Stimulation with irradiated EBV-transformed cells had a different impact on the activity of low- and high-density lymphocytes. Proliferative and cytotoxic responses were inversely correlated, i.e. high-density cells proliferated but exerted low cytotoxicity, while the lytic activity of the low density subset was stronger in the absence of significant cell proliferation. Proliferation and generation of cytotoxicity were abrogated by CsA in both subsets. The activities could be restored by addition of exogenous IL-2, suggesting that the drug may interfere with the cascade of lymphokine–cell interactions which leads to activation of immune responses. From the analysis of the CsA effects on the two subsets it seems that the high-density one contains specific memory T cells which are activated and proliferate upon encounter with EBV-infected cells. On the other hand, low-density lymphocytes are induced to release soluble factors with antiproliferative activity. The secretory function was resistant to the suppressive effect of CsA.
Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that is involved in the pathogenesis o... more Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that is involved in the pathogenesis of a wide spectrum of malignant and non-malignant diseases. Strong evidence implicates T lymphocytes in the control of EBV replication and tumorigenesis, but cellular components of the innate immune system are poorly characterized in terms of their function in the development of EBV-specific immunity or interaction with the virus. This study demonstrates that EBV virions produced in epithelial cells surpass their B cell-derived counterparts in the capacity to enter monocytes and inhibit their development into dendritic cells (DCs). Different ratios of the gp42 and gH glycoproteins in the envelope of virions that were derived from major histocompatibility complex class II-positive or -negative cells accounted primarily for the differences in EBV tropism. EBV is shown to enter both monocytes and DCs, although the cells are susceptible to virus-induced apoptosis only if infected at early stages of DC differentiation. The purified gH/gL heterodimer binds efficiently to monocytes and DCs, but not to B cells, suggesting that high expression levels of a putative binding partner for gH contribute to virus entry. This entry takes place despite very low or undetectable expression of CD21, the canonical EBV receptor. These results indicate that the site of virus replication, either in B cells or epithelial cells, alters EBV tropism for monocytes and DCs. This results in a change in the virus's immunomodulating capacity and may have important implications for the regulation of virus-host interactions during primary and chronic EBV infection.
We tested the effect of transforming growth factor (TGF)-beta 1 and TGF-beta 2 on the proliferati... more We tested the effect of transforming growth factor (TGF)-beta 1 and TGF-beta 2 on the proliferation of human B cell lines. The panel was selected to give information whether (1) their origin, (2) their phenotype, (3) their Epstein-Barr virus (EBV) carrier state, influence their responsiveness. The growth of lymphoblastoid cell lines (LCL) was not inhibited by TGF-beta 1. The EBV-carrying Burkitt lymphoma (BL) lines, Daudi, Jijoye, Rael but not Raji were inhibited. Three EBV-negative BL lines and the majority of their converted sublines were sensitive. The cell lines tested expressed TGF-beta receptors and TGF-beta 1 transcripts. The proliferation of EBV-infected B cells was inhibited by TGF-beta, their sensitivity decreased, however, after 3 days. The results suggest that the activation state of the B cells is decisive for TGF-beta sensitivity and EBV influences it indirectly by changing the cell phenotype.
Although malaria and Epstein-Barr (EBV) infection are recognized cofactors in the genesis of ende... more Although malaria and Epstein-Barr (EBV) infection are recognized cofactors in the genesis of endemic Burkitt lymphoma (BL), their relative contribution is not understood. BL, the most common paediatric cancer in equatorial Africa, is a high-grade B cell lymphoma characterized by c-myc translocation. EBV is a ubiquitous B lymphotropic virus that persists in a latent state after primary infection, and in Africa, most children have sero-converted by 3 y of age. Malaria infection profoundly affects the B cell compartment, inducing polyclonal activation and hyper-gammaglobulinemia. We recently identified the cystein-rich inter-domain region 1a (CIDR1a) of the Plasmodium falciparum membrane protein 1 as a polyclonal B cell activator that preferentially activates the memory compartment, where EBV is known to persist. Here, we have addressed the mechanisms of interaction between CIDR1a and EBV in the context of B cells. We show that CIDR1a binds to the EBV-positive B cell line Akata and increases the number of cells switching to the viral lytic cycle as measured by green fluorescent protein (GFP) expression driven by a lytic promoter. The virus production in CIDR1a-exposed cultures was directly proportional to the number of GFP-positive Akata cells (lytic EBV) and to the increased expression of the EBV lytic promoter BZLF1. Furthermore, CIDR1a stimulated the production of EBV in peripheral blood mononuclear cells derived from healthy donors and children with BL. Our results suggest that P. falciparum antigens such as CIDR1a can directly induce EBV reactivation during malaria infection that may increase the risk of BL development for children living in malaria-endemic areas. To our knowledge, this is the first report to show that a microbial protein can drive a latently infected B cell into EBV replication. Citation: Chêne A, Donati D, Guerreiro-Cacais AO, Levitsky V, Chen Q, et al. (2007) A molecular link between malaria and Epstein-Barr virus reactivation. PLoS Pathog 3(6): e80.
We have analyzed the EBV-related immune parameters of a healthy EBV-seropositive individual (ST) ... more We have analyzed the EBV-related immune parameters of a healthy EBV-seropositive individual (ST) who has regular antibody titers but defective inhibitory capacity toward the growth of autologous EBV-infected B cells. This in vitro function reflects the EBV-specific memory because it does not occur in experiments performed with cells of seronegative individuals. An analysis of events following in vitro EBV infection showed that lymphocytes of ST behaved in some tests in the same way as those collected from seronegative individuals. These parameters were: (I) lack of-γ-IFN production 24 hr after EBV infection; (2) low production of soluble factors that inhibit EBV-induced B-cell proliferation; (3) lack of generation of LCL selective cytotoxicity after repeated stimulation with autologous LCL; and (4) high proportion of EBNA-positive cells in 7-day-old EBV-infected cultures. On the other hand, cellular memory to the virus detected by the production of IL–2 24 hr after infection, and by the production of LIF upon exposure to EBV-encoded antigens, conformed with the results obtained with seropositive individuals. T-cell-mediated inhibition of EBV-induced B-cell growth in vitro has been regarded as a corollary of in vivo control of EBV-infected B cells. However, it is absent or has a low efficiency in certain disease categories which are not accompanied by risk of B-EBV growth. Our results with a healthy individual also indicate that several mechanisms contribute to a harmless lifelong virus carrier state.
Background The expression of recombinant proteins in Escherichia coli is an important and frequen... more Background The expression of recombinant proteins in Escherichia coli is an important and frequently used tool within malaria research, however, this method remains problematic. High A/T versus C/G content and frequent lysine and arginine repeats in the Plasmodium falciparum genome are thought to be the main reason for early termination in the mRNA translation process. Therefore, the majority of P. falciparum derived recombinant proteins is expressed only as truncated forms or appears as insoluble inclusion bodies within the bacterial cells. Methods Several domains of PfEMP1 genes obtained from different P. falciparum strains were expressed in E. coli as GST-fusion proteins. Expression was carried out under various culture conditions with a main focus on the time point of induction in relation to the bacterial growth stage. Results and conclusions When expressed in E. coli recombinant proteins derived from P. falciparum sequences are often truncated and tend to aggregate what in turn leads to the formation of insoluble inclusion bodies. The analysis of various factors influencing the expression revealed that the time point of induction plays a key role in successful expression of A/T rich sequences into their native conformation. Contrary to recommended procedures, initiation of expression at post-log instead of mid-log growth phase generated significantly increased amounts of soluble protein of a high quality. Furthermore, these proteins were shown to be functionally active. Other factors such as temperature, pH, bacterial proteases or the codon optimization for E. coli had little or no effect on the quality of the recombinant protein, nevertheless, optimizing these factors might be beneficial for each individual construct. In conclusion, changing the timepoint of induction and conducting expression at the post-log stage where the bacteria have entered a decelerated growth phase, greatly facilitates and improves the expression of sequences containing rare codons.
... 7.Fernandez, V., Hommel, M., Chen, Q., Hagblom, P. & Wahlgren, M. J. Exp ... Top of page.... more ... 7.Fernandez, V., Hommel, M., Chen, Q., Hagblom, P. & Wahlgren, M. J. Exp ... Top of page. Mats Wahlgren and Maria Teresa Bejarano are at the Microbiology and Tumour Biology Centre, Karolinska Institutet, and the Swedish Institute for Infectious Disease Control, Box 280, S-171 ...
Polyclonal B-cell activation and hypergammaglobulinemia are prominent features of human malaria. ... more Polyclonal B-cell activation and hypergammaglobulinemia are prominent features of human malaria. We report here that Plasmodium falciparum-infected erythrocytes directly adhere to and activate peripheral blood B cells from nonimmune donors. The infected erythrocytes employ the cysteine-rich interdomain region 1␣ (CIDR1␣) of P. falciparum erythrocyte membrane protein 1 (PfEMP1) to interact with the B cells. Stimulation with recombinant CIDR1␣ induces proliferation, an increase in B-cell size, expression of activation molecules, and secretion of immunoglobulins (immunoglobulin M) and cytokines (tumor necrosis factor alpha and interleukin-6). Furthermore, CIDR1␣ binds to Fab and Fc fragments of human immunoglobulins and to immunoglobulins purified from the sera of different animal species. This binding pattern is similar to that of the polyclonal B-cell activator Staphylococcus aureus protein A. Our findings shed light on the understanding of the molecular basis of polyclonal B-cell activation during malaria infections. The results suggest that the var gene family encoding PfEMP1 has evolved not only to mediate the sequestration of infected erythrocytes but also to manipulate the immune system to enhance the survival of the parasite.
We tested the effect of transforming growth factor (TGF)-beta 1 and TGF-beta 2 on the proliferati... more We tested the effect of transforming growth factor (TGF)-beta 1 and TGF-beta 2 on the proliferation of human B cell lines. The panel was selected to give information whether (1) their origin, (2) their phenotype, (3) their Epstein-Barr virus (EBV) carrier state, influence their responsiveness. The growth of lymphoblastoid cell lines (LCL) was not inhibited by TGF-beta 1. The EBV-carrying Burkitt lymphoma (BL) lines, Daudi, Jijoye, Rael but not Raji were inhibited. Three EBV-negative BL lines and the majority of their converted sublines were sensitive. The cell lines tested expressed TGF-beta receptors and TGF-beta 1 transcripts. The proliferation of EBV-infected B cells was inhibited by TGF-beta, their sensitivity decreased, however, after 3 days. The results suggest that the activation state of the B cells is decisive for TGF-beta sensitivity and EBV influences it indirectly by changing the cell phenotype.
Although malaria and Epstein-Barr (EBV) infection are recognized cofactors in the genesis of ende... more Although malaria and Epstein-Barr (EBV) infection are recognized cofactors in the genesis of endemic Burkitt lymphoma (BL), their relative contribution is not understood. BL, the most common paediatric cancer in equatorial Africa, is a high-grade B cell lymphoma characterized by c-myc translocation. EBV is a ubiquitous B lymphotropic virus that persists in a latent state after primary infection, and in Africa, most children have sero-converted by 3 y of age. Malaria infection profoundly affects the B cell compartment, inducing polyclonal activation and hyper-gammaglobulinemia. We recently identified the cystein-rich inter-domain region 1a (CIDR1a) of the Plasmodium falciparum membrane protein 1 as a polyclonal B cell activator that preferentially activates the memory compartment, where EBV is known to persist. Here, we have addressed the mechanisms of interaction between CIDR1a and EBV in the context of B cells. We show that CIDR1a binds to the EBV-positive B cell line Akata and increases the number of cells switching to the viral lytic cycle as measured by green fluorescent protein (GFP) expression driven by a lytic promoter. The virus production in CIDR1a-exposed cultures was directly proportional to the number of GFP-positive Akata cells (lytic EBV) and to the increased expression of the EBV lytic promoter BZLF1. Furthermore, CIDR1a stimulated the production of EBV in peripheral blood mononuclear cells derived from healthy donors and children with BL. Our results suggest that P. falciparum antigens such as CIDR1a can directly induce EBV reactivation during malaria infection that may increase the risk of BL development for children living in malaria-endemic areas. To our knowledge, this is the first report to show that a microbial protein can drive a latently infected B cell into EBV replication. Citation: Chêne A, Donati D, Guerreiro-Cacais AO, Levitsky V, Chen Q, et al. (2007) A molecular link between malaria and Epstein-Barr virus reactivation. PLoS Pathog 3(6): e80.
We have analyzed the EBV-related immune parameters of a healthy EBV-seropositive individual (ST) ... more We have analyzed the EBV-related immune parameters of a healthy EBV-seropositive individual (ST) who has regular antibody titers but defective inhibitory capacity toward the growth of autologous EBV-infected B cells. This in vitro function reflects the EBV-specific memory because it does not occur in experiments performed with cells of seronegative individuals. An analysis of events following in vitro EBV infection showed that lymphocytes of ST behaved in some tests in the same way as those collected from seronegative individuals. These parameters were: (I) lack of-γ-IFN production 24 hr after EBV infection; (2) low production of soluble factors that inhibit EBV-induced B-cell proliferation; (3) lack of generation of LCL selective cytotoxicity after repeated stimulation with autologous LCL; and (4) high proportion of EBNA-positive cells in 7-day-old EBV-infected cultures. On the other hand, cellular memory to the virus detected by the production of IL–2 24 hr after infection, and by the production of LIF upon exposure to EBV-encoded antigens, conformed with the results obtained with seropositive individuals. T-cell-mediated inhibition of EBV-induced B-cell growth in vitro has been regarded as a corollary of in vivo control of EBV-infected B cells. However, it is absent or has a low efficiency in certain disease categories which are not accompanied by risk of B-EBV growth. Our results with a healthy individual also indicate that several mechanisms contribute to a harmless lifelong virus carrier state.
Background The expression of recombinant proteins in Escherichia coli is an important and frequen... more Background The expression of recombinant proteins in Escherichia coli is an important and frequently used tool within malaria research, however, this method remains problematic. High A/T versus C/G content and frequent lysine and arginine repeats in the Plasmodium falciparum genome are thought to be the main reason for early termination in the mRNA translation process. Therefore, the majority of P. falciparum derived recombinant proteins is expressed only as truncated forms or appears as insoluble inclusion bodies within the bacterial cells. Methods Several domains of PfEMP1 genes obtained from different P. falciparum strains were expressed in E. coli as GST-fusion proteins. Expression was carried out under various culture conditions with a main focus on the time point of induction in relation to the bacterial growth stage. Results and conclusions When expressed in E. coli recombinant proteins derived from P. falciparum sequences are often truncated and tend to aggregate what in turn leads to the formation of insoluble inclusion bodies. The analysis of various factors influencing the expression revealed that the time point of induction plays a key role in successful expression of A/T rich sequences into their native conformation. Contrary to recommended procedures, initiation of expression at post-log instead of mid-log growth phase generated significantly increased amounts of soluble protein of a high quality. Furthermore, these proteins were shown to be functionally active. Other factors such as temperature, pH, bacterial proteases or the codon optimization for E. coli had little or no effect on the quality of the recombinant protein, nevertheless, optimizing these factors might be beneficial for each individual construct. In conclusion, changing the timepoint of induction and conducting expression at the post-log stage where the bacteria have entered a decelerated growth phase, greatly facilitates and improves the expression of sequences containing rare codons.
... 7.Fernandez, V., Hommel, M., Chen, Q., Hagblom, P. & Wahlgren, M. J. Exp ... Top of page.... more ... 7.Fernandez, V., Hommel, M., Chen, Q., Hagblom, P. & Wahlgren, M. J. Exp ... Top of page. Mats Wahlgren and Maria Teresa Bejarano are at the Microbiology and Tumour Biology Centre, Karolinska Institutet, and the Swedish Institute for Infectious Disease Control, Box 280, S-171 ...
Polyclonal B-cell activation and hypergammaglobulinemia are prominent features of human malaria. ... more Polyclonal B-cell activation and hypergammaglobulinemia are prominent features of human malaria. We report here that Plasmodium falciparum-infected erythrocytes directly adhere to and activate peripheral blood B cells from nonimmune donors. The infected erythrocytes employ the cysteine-rich interdomain region 1␣ (CIDR1␣) of P. falciparum erythrocyte membrane protein 1 (PfEMP1) to interact with the B cells. Stimulation with recombinant CIDR1␣ induces proliferation, an increase in B-cell size, expression of activation molecules, and secretion of immunoglobulins (immunoglobulin M) and cytokines (tumor necrosis factor alpha and interleukin-6). Furthermore, CIDR1␣ binds to Fab and Fc fragments of human immunoglobulins and to immunoglobulins purified from the sera of different animal species. This binding pattern is similar to that of the polyclonal B-cell activator Staphylococcus aureus protein A. Our findings shed light on the understanding of the molecular basis of polyclonal B-cell activation during malaria infections. The results suggest that the var gene family encoding PfEMP1 has evolved not only to mediate the sequestration of infected erythrocytes but also to manipulate the immune system to enhance the survival of the parasite.
Epstein-Barr-virus (EBV)-positive Burkitt's-lymphoma (BL) cell lines are not recognized by EBV-sp... more Epstein-Barr-virus (EBV)-positive Burkitt's-lymphoma (BL) cell lines are not recognized by EBV-specific T cells, due to their non-immunogenic phenotype and restricted expression of latent EBV genes. We tested whether triggering of CD40 can alter the phenotype of the tumor cells with regard to: (i) expression of surface markers, (ii) expression of viral antigens, (iii) presentation of endogenous antigens to MHCclass-I restricted cytotoxic T lymphocytes (CTLs), (iv) stimulatory capacity in allogeneic mixed-lymphocyte cultures (MLCs), (v) sensitivity to natural-killer (NK)-cell-mediated lysis. Co-culture of EBV-positive BL cells with CD40-ligandtransfected L cells induced up-regulation of CD54 and CD80 but did not affect the expression of viral genes. In spite of significant up-regulation of TAP1 and TAP2, and increased expression of MHC class I, the BL cells remained unable to present endogenously expressed viral antigens to EBVspecific CTL. However, the up-regulation of adhesion and co-stimulatory molecules was associated with increased stimulatory capacity in MLC and enhanced sensitivity to NK cells. These findings indicate that, while inducing only a modest phenotype shift, cross-linking of CD40 under physiologic conditions may selectively enhance the sensitivity of BL cells to anti-tumor immune responses. Int. J. Cancer 83:772-779, 1999.
Blood lymphocyte subsets of various densities from 14 healthy donors and 25 patients with solid t... more Blood lymphocyte subsets of various densities from 14 healthy donors and 25 patients with solid tumors were used in tests for cytotoxicity against allogeneic or autologous tumor cells. The allogeneic combinations comprised 36 tests on 28 tumors. In these the unmanipulated total lymphocyte population was cytotoxic in one experiment. In tests with density-separated subsets, 16 experiments were positive. The lytic effect resided in the light subsets. Auto-tumor lysis was detected in 7 of 25 cases, when the total lymphocyte population was used. Eleven additional tests showed cytotoxocity with the separated subsets. The autologous tumor cells were damaged by the light lymphocytes of 15 patients. Of these patients, 6 had cytotoxic cells also in the high-density subset. In seven cases auto-tumor lysis was exerted only by the dense lymphocytes. Thus, in 13 cases the profile of auto-tumor lysis differed from that observed with the allogeneic combinations and against K562. Pretreatment of the effectors with Hu-IFN-alpha enhanced or induced cytotoxicity in the light lymphocytes, against both allogeneic and autologous tumors, while the dense subset was rarely potentiated. The autologous system may represent an immune situation and therefore it is not unexpected that the active cells should have different phenotypic characteristics from those of the operationally natural killer system.
We have analyzed the effect of Cyclosporin-A (CsA) on the in vitro suppression of Epstein-Barr vi... more We have analyzed the effect of Cyclosporin-A (CsA) on the in vitro suppression of Epstein-Barr virus (EBV)-induced B-cell proliferation exerted by autologous T-lymphocyte subpopulations separated on the basis of cell density. CsA abolished the growth-inhibitory capacity of high-density T cells but influenced only marginally the activity of low-density lymphocytes; this suggested that different mechanisms mediate suppression in the two subsets. Stimulation with irradiated EBV-transformed cells had a different impact on the activity of low- and high-density lymphocytes. Proliferative and cytotoxic responses were inversely correlated, i.e. high-density cells proliferated but exerted low cytotoxicity, while the lytic activity of the low density subset was stronger in the absence of significant cell proliferation. Proliferation and generation of cytotoxicity were abrogated by CsA in both subsets. The activities could be restored by addition of exogenous IL-2, suggesting that the drug may interfere with the cascade of lymphokine–cell interactions which leads to activation of immune responses. From the analysis of the CsA effects on the two subsets it seems that the high-density one contains specific memory T cells which are activated and proliferate upon encounter with EBV-infected cells. On the other hand, low-density lymphocytes are induced to release soluble factors with antiproliferative activity. The secretory function was resistant to the suppressive effect of CsA.
Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that is involved in the pathogenesis o... more Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that is involved in the pathogenesis of a wide spectrum of malignant and non-malignant diseases. Strong evidence implicates T lymphocytes in the control of EBV replication and tumorigenesis, but cellular components of the innate immune system are poorly characterized in terms of their function in the development of EBV-specific immunity or interaction with the virus. This study demonstrates that EBV virions produced in epithelial cells surpass their B cell-derived counterparts in the capacity to enter monocytes and inhibit their development into dendritic cells (DCs). Different ratios of the gp42 and gH glycoproteins in the envelope of virions that were derived from major histocompatibility complex class II-positive or -negative cells accounted primarily for the differences in EBV tropism. EBV is shown to enter both monocytes and DCs, although the cells are susceptible to virus-induced apoptosis only if infected at early stages of DC differentiation. The purified gH/gL heterodimer binds efficiently to monocytes and DCs, but not to B cells, suggesting that high expression levels of a putative binding partner for gH contribute to virus entry. This entry takes place despite very low or undetectable expression of CD21, the canonical EBV receptor. These results indicate that the site of virus replication, either in B cells or epithelial cells, alters EBV tropism for monocytes and DCs. This results in a change in the virus's immunomodulating capacity and may have important implications for the regulation of virus-host interactions during primary and chronic EBV infection.
We tested the effect of transforming growth factor (TGF)-beta 1 and TGF-beta 2 on the proliferati... more We tested the effect of transforming growth factor (TGF)-beta 1 and TGF-beta 2 on the proliferation of human B cell lines. The panel was selected to give information whether (1) their origin, (2) their phenotype, (3) their Epstein-Barr virus (EBV) carrier state, influence their responsiveness. The growth of lymphoblastoid cell lines (LCL) was not inhibited by TGF-beta 1. The EBV-carrying Burkitt lymphoma (BL) lines, Daudi, Jijoye, Rael but not Raji were inhibited. Three EBV-negative BL lines and the majority of their converted sublines were sensitive. The cell lines tested expressed TGF-beta receptors and TGF-beta 1 transcripts. The proliferation of EBV-infected B cells was inhibited by TGF-beta, their sensitivity decreased, however, after 3 days. The results suggest that the activation state of the B cells is decisive for TGF-beta sensitivity and EBV influences it indirectly by changing the cell phenotype.
Although malaria and Epstein-Barr (EBV) infection are recognized cofactors in the genesis of ende... more Although malaria and Epstein-Barr (EBV) infection are recognized cofactors in the genesis of endemic Burkitt lymphoma (BL), their relative contribution is not understood. BL, the most common paediatric cancer in equatorial Africa, is a high-grade B cell lymphoma characterized by c-myc translocation. EBV is a ubiquitous B lymphotropic virus that persists in a latent state after primary infection, and in Africa, most children have sero-converted by 3 y of age. Malaria infection profoundly affects the B cell compartment, inducing polyclonal activation and hyper-gammaglobulinemia. We recently identified the cystein-rich inter-domain region 1a (CIDR1a) of the Plasmodium falciparum membrane protein 1 as a polyclonal B cell activator that preferentially activates the memory compartment, where EBV is known to persist. Here, we have addressed the mechanisms of interaction between CIDR1a and EBV in the context of B cells. We show that CIDR1a binds to the EBV-positive B cell line Akata and increases the number of cells switching to the viral lytic cycle as measured by green fluorescent protein (GFP) expression driven by a lytic promoter. The virus production in CIDR1a-exposed cultures was directly proportional to the number of GFP-positive Akata cells (lytic EBV) and to the increased expression of the EBV lytic promoter BZLF1. Furthermore, CIDR1a stimulated the production of EBV in peripheral blood mononuclear cells derived from healthy donors and children with BL. Our results suggest that P. falciparum antigens such as CIDR1a can directly induce EBV reactivation during malaria infection that may increase the risk of BL development for children living in malaria-endemic areas. To our knowledge, this is the first report to show that a microbial protein can drive a latently infected B cell into EBV replication. Citation: Chêne A, Donati D, Guerreiro-Cacais AO, Levitsky V, Chen Q, et al. (2007) A molecular link between malaria and Epstein-Barr virus reactivation. PLoS Pathog 3(6): e80.
We have analyzed the EBV-related immune parameters of a healthy EBV-seropositive individual (ST) ... more We have analyzed the EBV-related immune parameters of a healthy EBV-seropositive individual (ST) who has regular antibody titers but defective inhibitory capacity toward the growth of autologous EBV-infected B cells. This in vitro function reflects the EBV-specific memory because it does not occur in experiments performed with cells of seronegative individuals. An analysis of events following in vitro EBV infection showed that lymphocytes of ST behaved in some tests in the same way as those collected from seronegative individuals. These parameters were: (I) lack of-γ-IFN production 24 hr after EBV infection; (2) low production of soluble factors that inhibit EBV-induced B-cell proliferation; (3) lack of generation of LCL selective cytotoxicity after repeated stimulation with autologous LCL; and (4) high proportion of EBNA-positive cells in 7-day-old EBV-infected cultures. On the other hand, cellular memory to the virus detected by the production of IL–2 24 hr after infection, and by the production of LIF upon exposure to EBV-encoded antigens, conformed with the results obtained with seropositive individuals. T-cell-mediated inhibition of EBV-induced B-cell growth in vitro has been regarded as a corollary of in vivo control of EBV-infected B cells. However, it is absent or has a low efficiency in certain disease categories which are not accompanied by risk of B-EBV growth. Our results with a healthy individual also indicate that several mechanisms contribute to a harmless lifelong virus carrier state.
Background The expression of recombinant proteins in Escherichia coli is an important and frequen... more Background The expression of recombinant proteins in Escherichia coli is an important and frequently used tool within malaria research, however, this method remains problematic. High A/T versus C/G content and frequent lysine and arginine repeats in the Plasmodium falciparum genome are thought to be the main reason for early termination in the mRNA translation process. Therefore, the majority of P. falciparum derived recombinant proteins is expressed only as truncated forms or appears as insoluble inclusion bodies within the bacterial cells. Methods Several domains of PfEMP1 genes obtained from different P. falciparum strains were expressed in E. coli as GST-fusion proteins. Expression was carried out under various culture conditions with a main focus on the time point of induction in relation to the bacterial growth stage. Results and conclusions When expressed in E. coli recombinant proteins derived from P. falciparum sequences are often truncated and tend to aggregate what in turn leads to the formation of insoluble inclusion bodies. The analysis of various factors influencing the expression revealed that the time point of induction plays a key role in successful expression of A/T rich sequences into their native conformation. Contrary to recommended procedures, initiation of expression at post-log instead of mid-log growth phase generated significantly increased amounts of soluble protein of a high quality. Furthermore, these proteins were shown to be functionally active. Other factors such as temperature, pH, bacterial proteases or the codon optimization for E. coli had little or no effect on the quality of the recombinant protein, nevertheless, optimizing these factors might be beneficial for each individual construct. In conclusion, changing the timepoint of induction and conducting expression at the post-log stage where the bacteria have entered a decelerated growth phase, greatly facilitates and improves the expression of sequences containing rare codons.
... 7.Fernandez, V., Hommel, M., Chen, Q., Hagblom, P. & Wahlgren, M. J. Exp ... Top of page.... more ... 7.Fernandez, V., Hommel, M., Chen, Q., Hagblom, P. & Wahlgren, M. J. Exp ... Top of page. Mats Wahlgren and Maria Teresa Bejarano are at the Microbiology and Tumour Biology Centre, Karolinska Institutet, and the Swedish Institute for Infectious Disease Control, Box 280, S-171 ...
Polyclonal B-cell activation and hypergammaglobulinemia are prominent features of human malaria. ... more Polyclonal B-cell activation and hypergammaglobulinemia are prominent features of human malaria. We report here that Plasmodium falciparum-infected erythrocytes directly adhere to and activate peripheral blood B cells from nonimmune donors. The infected erythrocytes employ the cysteine-rich interdomain region 1␣ (CIDR1␣) of P. falciparum erythrocyte membrane protein 1 (PfEMP1) to interact with the B cells. Stimulation with recombinant CIDR1␣ induces proliferation, an increase in B-cell size, expression of activation molecules, and secretion of immunoglobulins (immunoglobulin M) and cytokines (tumor necrosis factor alpha and interleukin-6). Furthermore, CIDR1␣ binds to Fab and Fc fragments of human immunoglobulins and to immunoglobulins purified from the sera of different animal species. This binding pattern is similar to that of the polyclonal B-cell activator Staphylococcus aureus protein A. Our findings shed light on the understanding of the molecular basis of polyclonal B-cell activation during malaria infections. The results suggest that the var gene family encoding PfEMP1 has evolved not only to mediate the sequestration of infected erythrocytes but also to manipulate the immune system to enhance the survival of the parasite.
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Papers by Maria Bejarano