Virus Research 195 (2015) 95–99
Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Elevated antinuclear antibodies and altered anti-Epstein-Barr virus immune responses Laura Cuomo a , Mara Cirone d , Ana Oliva Di Gregorio a , Marina Vitillo a , Marina Cattivelli b , Vittoria Magliocca a , Silvana Maiorano b , Marcello Meledandri b , Carolina Scagnolari c , Sebastiano La Rocca a , Pankaj Trivedi d,∗ a
U.O.C. Patologia Clinica, A.C.O. San Filippo Neri, Rome, Italy U.O.C. Microbiologia e Virologia, A.C.O. San Filippo Neri, Rome, Italy c Department of Molecular Medicine, La Sapienza University, Rome, Italy d Department of Experimental Medicine, La Sapienza University, Rome, Italy b
a r t i c l e
i n f o
Article history: Received 4 June 2014 Received in revised form 28 September 2014 Accepted 29 September 2014 Available online 7 October 2014 Keywords: EBV Autoimmunity EBNA1 ANA SLE VCA
a b s t r a c t It has been shown that Epstein-Barr virus (EBV) is able to alter the immune response towards self-antigens and may enhance risk of autoimmune diseases such as systemic lupus erythematosus (SLE) in genetically predisposed individuals. In this study, we evaluated the specific antibody immune response against EBV in patients with anti-nuclear autoantibodies (ANA) in comparison with ANA-negative healthy controls. For this purpose, 92 patients with an high anti-ANA reactivity with or without concomitant extractable nuclear antigen (ENA) or double stranded DNA (dsDNA) positivity were selected and compared with 146 healthy donors. We found that anti-EBV-VCA and EA IgG concentrations were significantly higher in ANA-positive patients in comparison to the controls (VCA P < 0.0001 and EA P < 0,03) as well as in those ANA-positive patients that showed a concomitant ENA positivity (P = 0.0002). Interestingly, elevated antiEBNA-1 IgG was found in a group of patients who had anti SSA/Ro antibodies. Anti-VCA IgM Abs were more frequently found in those patients with a very high titer of ANA (P = 0.06); moreover detection of anti-VCA IgM/IgG in absence of anti-EBNA-1 IgG was more frequent in the patient than in the control group. Both these conditions correlate with a recent EBV infection or reactivation. The data suggest that EBV, particularly during acute infection or in its reactivation phase, could be involved in the ANA and ENA autoantibody formation. © 2014 Elsevier B.V. All rights reserved.
1. Introduction EBV is a ubiquitous gamma herpesvirus which infects more than 90% of the human population worldwide (Klein et al., 2007). The virus establishes a latent infection in B cells by expressing only a limited number of viral antigens (Babcock et al., 2000). The number of infected cells remains generally constant over a lifetime in healthy individuals with occasional flare in viral replication mainly related to B cell homeostasis and developmental cues received by the infected cells. During acute infection, which causes infectious mononucleosis (IM) in 50% of the young adults, a massive viral replication occurs. It is estimated that IM patients have 20 fold increased risk of developing multiple sclerosis and
∗ Corresponding author. Tel.: +39 06 49973015. E-mail address: [email protected] (P. Trivedi). http://dx.doi.org/10.1016/j.virusres.2014.09.014 0168-1702/© 2014 Elsevier B.V. All rights reserved.
other autoimmune diseases (Ascherio and Munger, 2007; James et al., 2001). While the link between EBV and lymphomagenesis is becoming increasingly clearer, the contribution of the virus in autoimmunity still remains fairly obscure. It is suspected that deregulated virus-specific immune response could underlie autoimmune diseases (Poole et al., 2009). The development of autoimmune diseases such as Systemic Lupus Erythematosus (SLE) seems to be associated with both genetic and environmental factors (Klein-Gitelman and Miller, 2004). In the former category, HLA-DR and HLA-DQ alleles strongly predispose to SLE development whereas in the latter category, EBV, cytomegalovirus (CMV) and human endogenous retroviruses (HERVs) have been proposed to be involved (Klein-Gitelman and Miller, 2004). The role of EBV in triggering autoimmune diseases has become evident first from serological and subsequently from in situ studies in maladies like SLE, RA and multiple sclerosis (Denman, 2000; James and Robertson, 2012; Niller et al., 2008). As far as
96
L. Cuomo et al. / Virus Research 195 (2015) 95–99
SLE is concerned, the serological studies initially indicated that almost 100% of SLE patients are EBV seropositive against about 90% seropositivity in healthy population, characterized by higher VCAIgG (James et al., 1997a). Furthermore, increased viral load in blood and saliva are frequent (Moon et al., 2004; Strauch et al., 1974). These data are further backed by DNA analysis in SLE patients (Yu et al., 2005). The increased EBV load described in SLE patients could be dependent on an increased replication or a higher number of latenly infected B cells. Recently, Gross et al. (2005) showed that SLE patients have higher number of cells carrying EBV in blood and that it is particularly high during the flares, typically observed in SLE. It was thus suggested that the immune system perturbation associated with SLE can be the cause of EBV spread and of the increased number of infected cells (Gross et al., 2005). The immunological dysfunction in SLE is correlated both to B and T cell abnormalities which result in production of autoantibodies to nuclear antigens generating immune complexes which consequently damage various organs. The B cell dysfunction in SLE creates auto-reactive B cell population which produces anti-Sm and anti SSA/Ro antibodies (Arbuckle et al., 2003). Interestingly, antibodies directed against Sm and Ro proteins cross-react with the EBV antigens EBNA-1 and EBNA-2, suggesting molecular mimicry as a potential mechanism (Sabbadini et al., 1993; Incaprera et al., 1998; McClain et al., 2005). The role of the crossreaction between antibodies anti-EBNA1 and autoantibodies to Sm and dsDNA in the onset of SLE is further strengthened by the observation that rabbits and mice immunized with EBNA1 DNA show symptoms of SLE (Poole et al., 2008; James et al., 1997b). Dysfunction of T cell responses in SLE has also been described. Specifically, a disruption in EBNA1 related antibodydependent cellular cytotoxicity and a reduced T cell cytotoxicity against EBV infected cells are noted in SLE (Rothfield et al., 1973; McClain et al., 2006). Additionally, EBV specific T cell cytotoxicity is also compromised in SLE patients (Berner et al., 2005). In this study, we have extended previous observations on altered EBV specific responses in SLE patients, to subjects with high ANA titers and various ENA positivity. Moreover, we found that a recent EBV infection or reactivation might have an important role in eliciting autoantibody deregulation. 2. Materials and methods 2.1. Patient population Ninety-two patients attending the outpatients’ department at our hospital were recruited for this study. Criteria for selection were anti-nuclear positive staining ≥ 1:160 and/or ENA/dsDNA positive detection (Table 1). The cut-off titer was chosen according to the existing guidelines which indicate that a titer over 1:160 could suggest the presence of an autoimmune disease (Kavanaugh et al., 2000). One hundred-forty-six healthy volunteers were recruited among the blood donors from the Hematology Department. All healthy donors were negative for anti-nuclear antibody. 2.2. Indirect Immunofluorescence (IFI) staining Anti-nuclear antibodies detection was performed by IFI using Hep-2 laryngeal carcinoma cells as substrate (Delta Biological). The dsDNA autoantibodies were detected on Crithidia Luciliae cells (Chematil) according to manufacturer instructions. The anti-ENA autoantibodies, U1RNP (A,B,C), Sm-D, Scl70, CENP B, Jo-1, SSA/Ro, SSB/La were detected in ELISA (Alifax) and the results were confirmed by immunoblotting (Euroimmun).
Table 1 ANA, ENA and dsDNA antibodies expression in 92 patients.a Number of patients (n = 92)
ANA-main fluorescence pattern
ENA positive (n = 57)
dsDNA positive (n = 10)
19
Anti-centromere
1
24
Homogeneous
3
6
Nucleolus organized region (NOR 90) Nucleolar
17 CENP B 1 CENPB/SSA/SSB 4 Scl70 3 SSA/Ro –
–
–
40
Speckled
13 SSA/Ro 11 SSA/Ro + SSB/La 5 RNP 1 Jo-1 1 SSA/Ro + RNP 1 SSA/Ro + Sm + RNP
1
a
8 –
Patients included in the study showed ANA titer ≥1:160.
2.3. EBV-specific antigen detection EBV-VCA IgM, -VCA IgG and -EBNA-1 IgG detection were performed by ELISA (Diasorin). EA IgG detection method is based on the ELISA principle and was performed using CHORUS kit and instrument (DIESSE). 2.4. Statistical analysis Statistical analysis was performed using the GraphPad software. The bee-swarm plots were prepared with the help of R software. The P value and the statistical significance were evaluated by unpaired t-test. The analysis between percentage of positive and negative groups was performed by Fischer’s exact test. 3. Results 3.1. Increased anti-VCA-IgG in ANA positive patients Ninety-two ANA positive patients with different reactivity patterns (Table 1) and 146 healthy blood donors were compared for EBV specific antibody response. We found that VCA-IgG was significantly higher in the ANA-positive patients group (P = 0.0001, Table 2, Fig. 1). A significant difference in VCA-IgG was also found when the analysis was restricted to patients with a concomitant ANA and ENA (n = 57) positivity (P = 0.0002) (Fig. 2). The ENA positive patients showed a various pattern of reactivity (Table 1). No individual from the control group showed any anti-nuclear reactivity. In contrast, the anti-VCA IgM and anti-EBNA-1 IgG antibody levels were not significantly different in the ANA positive and healthy control groups (Table 2). Additionally, the percentage of positive patients for these antibodies did not show significant differences among the two groups (Table 2). Among the ANA-positive subjects, we found a higher percentage of VCA-IgG and/or VCA IgM positive patients that were concomitantly negative for anti-EBNA-1 antibodies. The VCA IgM/IgG positivity associated with EBNA-1 negativity is indicative of a recent EBV infection (Table 2) (Klutts et al., 2009). 3.2. Increased EBNA1 IgG in SSA/Ro positive patients A high homology between SSA/Ro and EBNA-1 proteins has been previously shown (McClain et al., 2005). Based on this, we selected those patients positive for SSA/Ro antigen without any other ENA positivity and compared their antibody response against
L. Cuomo et al. / Virus Research 195 (2015) 95–99
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Table 2 Antibody concentration against EBV-specific antigens and percentage of positive subjects in ANA positive patients and controls. P value
EBV-specific Ab concentration (mean)a
ANA positive patients (n = 92)
Control (n = 146)
VCA IgM % Positived VCA IgG % Positived EBNA-1 IgG % Positived EA IgGb % Positived
0.291 5.4 12.279 96.7 5.518 81.5 0.735 (n = 52) 17.3
0.292 4.1 8.880 97.3 5.376 87.7 0.167 (n = 39) 2.5
0.997 0.753 0.0001 1.000 0.768 0.195 0.0235 0.0393
% VCA+/EBNA1−IgGc
17.3
9.5
0.106
a
Ab concentrations were expressed as an index (ratio between sample O.D. and cut-off value) as detected by ELISA. The ratio >1 is considered positive and it is proportional to the amount of specific Ig present. The p value < 0.05 is considered statistically significant. The statistically significant data is highlighted. b For EA IgG the ratio >1.2 is considered positive. c The figures indicate the percentage of subjects who were positive for VCA IgG and/or IgM but EBNA-1 IgG negative. d Percentage of individuals positive for each EBV antigen within the specified group.
Fig. 2. EBV-VCA IgG concentration in ENA positive individuals: The analysis was carried out in 57 ENA positive subjects (out of 92 ANA positive patients) in comparison with 146 ANA/ENA negative healthy donors. (P = 0.0002). The amount abs are shown as an index ratio between sample OD and the cut-off value. Mean values are indicated by bars.
EBNA-1 to that of patients positive for ENA other than SSA/Ro. As shown in Fig. 3, anti-EBNA-1 IgG was higher in SSA/Ro positive group (P = 0.07) while no differences were observed in anti-VCA IgG and anti-VCA IgM between the two groups (data not shown). These results are consistent with a previous report (McClain et al., 2005) that there is cross-reactivity in the specific antibody response towards EBNA-1 and SSA/Ro. 3.3. Correlation between EBV-VCA IgM and ANA titer ANA positive patients were divided in two groups based on their anti-nuclear antibodies titers where the cutoff titer was 1:640. We investigated anti-EBV specific antibody profile in these groups. Although chance cannot be excluded, we did find a significantly increased level of anti-VCA IgM in patients with high anti-nuclear antibodies (P = 0.06) (Fig. 4), suggesting that an EBV acute infection or reactivation might be associated with the anti-nuclear autoantibody formation. Fig. 3. EBV-EBNA-1 IgG levels in SSA/Ro positive subjects: Sixteen patients with positivity only for SSA/Ro were compared with 27 ENA positive but SSA/Ro negative individuals. All subjects were ANA positive. (P = 0.07).
Fig. 1. EBV-VCA-IgG concentration in ANA positive patients: The distribution of VCA IgG was evaluated in ELISA in 92 ANA positive patients in comparison with 146 ANA negative healthy subjects. (P < 0.0001). The bar indicates mean values. The Ab concentrations were expressed as an index (ratio between sample O.D. and cut-off value) as detected by ELISA. The ratio >1 is considered positive.
Fig. 4. Correlation between ANA titer and EBV-VCA-IgM concentration: 92 individuals were divided into two groups according to their ANA titer (
Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Elevated antinuclear antibodies and altered anti-Epstein-Barr virus immune responses Laura Cuomo a , Mara Cirone d , Ana Oliva Di Gregorio a , Marina Vitillo a , Marina Cattivelli b , Vittoria Magliocca a , Silvana Maiorano b , Marcello Meledandri b , Carolina Scagnolari c , Sebastiano La Rocca a , Pankaj Trivedi d,∗ a
U.O.C. Patologia Clinica, A.C.O. San Filippo Neri, Rome, Italy U.O.C. Microbiologia e Virologia, A.C.O. San Filippo Neri, Rome, Italy c Department of Molecular Medicine, La Sapienza University, Rome, Italy d Department of Experimental Medicine, La Sapienza University, Rome, Italy b
a r t i c l e
i n f o
Article history: Received 4 June 2014 Received in revised form 28 September 2014 Accepted 29 September 2014 Available online 7 October 2014 Keywords: EBV Autoimmunity EBNA1 ANA SLE VCA
a b s t r a c t It has been shown that Epstein-Barr virus (EBV) is able to alter the immune response towards self-antigens and may enhance risk of autoimmune diseases such as systemic lupus erythematosus (SLE) in genetically predisposed individuals. In this study, we evaluated the specific antibody immune response against EBV in patients with anti-nuclear autoantibodies (ANA) in comparison with ANA-negative healthy controls. For this purpose, 92 patients with an high anti-ANA reactivity with or without concomitant extractable nuclear antigen (ENA) or double stranded DNA (dsDNA) positivity were selected and compared with 146 healthy donors. We found that anti-EBV-VCA and EA IgG concentrations were significantly higher in ANA-positive patients in comparison to the controls (VCA P < 0.0001 and EA P < 0,03) as well as in those ANA-positive patients that showed a concomitant ENA positivity (P = 0.0002). Interestingly, elevated antiEBNA-1 IgG was found in a group of patients who had anti SSA/Ro antibodies. Anti-VCA IgM Abs were more frequently found in those patients with a very high titer of ANA (P = 0.06); moreover detection of anti-VCA IgM/IgG in absence of anti-EBNA-1 IgG was more frequent in the patient than in the control group. Both these conditions correlate with a recent EBV infection or reactivation. The data suggest that EBV, particularly during acute infection or in its reactivation phase, could be involved in the ANA and ENA autoantibody formation. © 2014 Elsevier B.V. All rights reserved.
1. Introduction EBV is a ubiquitous gamma herpesvirus which infects more than 90% of the human population worldwide (Klein et al., 2007). The virus establishes a latent infection in B cells by expressing only a limited number of viral antigens (Babcock et al., 2000). The number of infected cells remains generally constant over a lifetime in healthy individuals with occasional flare in viral replication mainly related to B cell homeostasis and developmental cues received by the infected cells. During acute infection, which causes infectious mononucleosis (IM) in 50% of the young adults, a massive viral replication occurs. It is estimated that IM patients have 20 fold increased risk of developing multiple sclerosis and
∗ Corresponding author. Tel.: +39 06 49973015. E-mail address: [email protected] (P. Trivedi). http://dx.doi.org/10.1016/j.virusres.2014.09.014 0168-1702/© 2014 Elsevier B.V. All rights reserved.
other autoimmune diseases (Ascherio and Munger, 2007; James et al., 2001). While the link between EBV and lymphomagenesis is becoming increasingly clearer, the contribution of the virus in autoimmunity still remains fairly obscure. It is suspected that deregulated virus-specific immune response could underlie autoimmune diseases (Poole et al., 2009). The development of autoimmune diseases such as Systemic Lupus Erythematosus (SLE) seems to be associated with both genetic and environmental factors (Klein-Gitelman and Miller, 2004). In the former category, HLA-DR and HLA-DQ alleles strongly predispose to SLE development whereas in the latter category, EBV, cytomegalovirus (CMV) and human endogenous retroviruses (HERVs) have been proposed to be involved (Klein-Gitelman and Miller, 2004). The role of EBV in triggering autoimmune diseases has become evident first from serological and subsequently from in situ studies in maladies like SLE, RA and multiple sclerosis (Denman, 2000; James and Robertson, 2012; Niller et al., 2008). As far as
96
L. Cuomo et al. / Virus Research 195 (2015) 95–99
SLE is concerned, the serological studies initially indicated that almost 100% of SLE patients are EBV seropositive against about 90% seropositivity in healthy population, characterized by higher VCAIgG (James et al., 1997a). Furthermore, increased viral load in blood and saliva are frequent (Moon et al., 2004; Strauch et al., 1974). These data are further backed by DNA analysis in SLE patients (Yu et al., 2005). The increased EBV load described in SLE patients could be dependent on an increased replication or a higher number of latenly infected B cells. Recently, Gross et al. (2005) showed that SLE patients have higher number of cells carrying EBV in blood and that it is particularly high during the flares, typically observed in SLE. It was thus suggested that the immune system perturbation associated with SLE can be the cause of EBV spread and of the increased number of infected cells (Gross et al., 2005). The immunological dysfunction in SLE is correlated both to B and T cell abnormalities which result in production of autoantibodies to nuclear antigens generating immune complexes which consequently damage various organs. The B cell dysfunction in SLE creates auto-reactive B cell population which produces anti-Sm and anti SSA/Ro antibodies (Arbuckle et al., 2003). Interestingly, antibodies directed against Sm and Ro proteins cross-react with the EBV antigens EBNA-1 and EBNA-2, suggesting molecular mimicry as a potential mechanism (Sabbadini et al., 1993; Incaprera et al., 1998; McClain et al., 2005). The role of the crossreaction between antibodies anti-EBNA1 and autoantibodies to Sm and dsDNA in the onset of SLE is further strengthened by the observation that rabbits and mice immunized with EBNA1 DNA show symptoms of SLE (Poole et al., 2008; James et al., 1997b). Dysfunction of T cell responses in SLE has also been described. Specifically, a disruption in EBNA1 related antibodydependent cellular cytotoxicity and a reduced T cell cytotoxicity against EBV infected cells are noted in SLE (Rothfield et al., 1973; McClain et al., 2006). Additionally, EBV specific T cell cytotoxicity is also compromised in SLE patients (Berner et al., 2005). In this study, we have extended previous observations on altered EBV specific responses in SLE patients, to subjects with high ANA titers and various ENA positivity. Moreover, we found that a recent EBV infection or reactivation might have an important role in eliciting autoantibody deregulation. 2. Materials and methods 2.1. Patient population Ninety-two patients attending the outpatients’ department at our hospital were recruited for this study. Criteria for selection were anti-nuclear positive staining ≥ 1:160 and/or ENA/dsDNA positive detection (Table 1). The cut-off titer was chosen according to the existing guidelines which indicate that a titer over 1:160 could suggest the presence of an autoimmune disease (Kavanaugh et al., 2000). One hundred-forty-six healthy volunteers were recruited among the blood donors from the Hematology Department. All healthy donors were negative for anti-nuclear antibody. 2.2. Indirect Immunofluorescence (IFI) staining Anti-nuclear antibodies detection was performed by IFI using Hep-2 laryngeal carcinoma cells as substrate (Delta Biological). The dsDNA autoantibodies were detected on Crithidia Luciliae cells (Chematil) according to manufacturer instructions. The anti-ENA autoantibodies, U1RNP (A,B,C), Sm-D, Scl70, CENP B, Jo-1, SSA/Ro, SSB/La were detected in ELISA (Alifax) and the results were confirmed by immunoblotting (Euroimmun).
Table 1 ANA, ENA and dsDNA antibodies expression in 92 patients.a Number of patients (n = 92)
ANA-main fluorescence pattern
ENA positive (n = 57)
dsDNA positive (n = 10)
19
Anti-centromere
1
24
Homogeneous
3
6
Nucleolus organized region (NOR 90) Nucleolar
17 CENP B 1 CENPB/SSA/SSB 4 Scl70 3 SSA/Ro –
–
–
40
Speckled
13 SSA/Ro 11 SSA/Ro + SSB/La 5 RNP 1 Jo-1 1 SSA/Ro + RNP 1 SSA/Ro + Sm + RNP
1
a
8 –
Patients included in the study showed ANA titer ≥1:160.
2.3. EBV-specific antigen detection EBV-VCA IgM, -VCA IgG and -EBNA-1 IgG detection were performed by ELISA (Diasorin). EA IgG detection method is based on the ELISA principle and was performed using CHORUS kit and instrument (DIESSE). 2.4. Statistical analysis Statistical analysis was performed using the GraphPad software. The bee-swarm plots were prepared with the help of R software. The P value and the statistical significance were evaluated by unpaired t-test. The analysis between percentage of positive and negative groups was performed by Fischer’s exact test. 3. Results 3.1. Increased anti-VCA-IgG in ANA positive patients Ninety-two ANA positive patients with different reactivity patterns (Table 1) and 146 healthy blood donors were compared for EBV specific antibody response. We found that VCA-IgG was significantly higher in the ANA-positive patients group (P = 0.0001, Table 2, Fig. 1). A significant difference in VCA-IgG was also found when the analysis was restricted to patients with a concomitant ANA and ENA (n = 57) positivity (P = 0.0002) (Fig. 2). The ENA positive patients showed a various pattern of reactivity (Table 1). No individual from the control group showed any anti-nuclear reactivity. In contrast, the anti-VCA IgM and anti-EBNA-1 IgG antibody levels were not significantly different in the ANA positive and healthy control groups (Table 2). Additionally, the percentage of positive patients for these antibodies did not show significant differences among the two groups (Table 2). Among the ANA-positive subjects, we found a higher percentage of VCA-IgG and/or VCA IgM positive patients that were concomitantly negative for anti-EBNA-1 antibodies. The VCA IgM/IgG positivity associated with EBNA-1 negativity is indicative of a recent EBV infection (Table 2) (Klutts et al., 2009). 3.2. Increased EBNA1 IgG in SSA/Ro positive patients A high homology between SSA/Ro and EBNA-1 proteins has been previously shown (McClain et al., 2005). Based on this, we selected those patients positive for SSA/Ro antigen without any other ENA positivity and compared their antibody response against
L. Cuomo et al. / Virus Research 195 (2015) 95–99
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Table 2 Antibody concentration against EBV-specific antigens and percentage of positive subjects in ANA positive patients and controls. P value
EBV-specific Ab concentration (mean)a
ANA positive patients (n = 92)
Control (n = 146)
VCA IgM % Positived VCA IgG % Positived EBNA-1 IgG % Positived EA IgGb % Positived
0.291 5.4 12.279 96.7 5.518 81.5 0.735 (n = 52) 17.3
0.292 4.1 8.880 97.3 5.376 87.7 0.167 (n = 39) 2.5
0.997 0.753 0.0001 1.000 0.768 0.195 0.0235 0.0393
% VCA+/EBNA1−IgGc
17.3
9.5
0.106
a
Ab concentrations were expressed as an index (ratio between sample O.D. and cut-off value) as detected by ELISA. The ratio >1 is considered positive and it is proportional to the amount of specific Ig present. The p value < 0.05 is considered statistically significant. The statistically significant data is highlighted. b For EA IgG the ratio >1.2 is considered positive. c The figures indicate the percentage of subjects who were positive for VCA IgG and/or IgM but EBNA-1 IgG negative. d Percentage of individuals positive for each EBV antigen within the specified group.
Fig. 2. EBV-VCA IgG concentration in ENA positive individuals: The analysis was carried out in 57 ENA positive subjects (out of 92 ANA positive patients) in comparison with 146 ANA/ENA negative healthy donors. (P = 0.0002). The amount abs are shown as an index ratio between sample OD and the cut-off value. Mean values are indicated by bars.
EBNA-1 to that of patients positive for ENA other than SSA/Ro. As shown in Fig. 3, anti-EBNA-1 IgG was higher in SSA/Ro positive group (P = 0.07) while no differences were observed in anti-VCA IgG and anti-VCA IgM between the two groups (data not shown). These results are consistent with a previous report (McClain et al., 2005) that there is cross-reactivity in the specific antibody response towards EBNA-1 and SSA/Ro. 3.3. Correlation between EBV-VCA IgM and ANA titer ANA positive patients were divided in two groups based on their anti-nuclear antibodies titers where the cutoff titer was 1:640. We investigated anti-EBV specific antibody profile in these groups. Although chance cannot be excluded, we did find a significantly increased level of anti-VCA IgM in patients with high anti-nuclear antibodies (P = 0.06) (Fig. 4), suggesting that an EBV acute infection or reactivation might be associated with the anti-nuclear autoantibody formation. Fig. 3. EBV-EBNA-1 IgG levels in SSA/Ro positive subjects: Sixteen patients with positivity only for SSA/Ro were compared with 27 ENA positive but SSA/Ro negative individuals. All subjects were ANA positive. (P = 0.07).
Fig. 1. EBV-VCA-IgG concentration in ANA positive patients: The distribution of VCA IgG was evaluated in ELISA in 92 ANA positive patients in comparison with 146 ANA negative healthy subjects. (P < 0.0001). The bar indicates mean values. The Ab concentrations were expressed as an index (ratio between sample O.D. and cut-off value) as detected by ELISA. The ratio >1 is considered positive.
Fig. 4. Correlation between ANA titer and EBV-VCA-IgM concentration: 92 individuals were divided into two groups according to their ANA titer (
