Epstein-Barr Virus Early Antigen Research Articles

Immunological Aspects of EBV and Oral Mucosa Interactions in Oral Lichen Planus

Oral lichen planus (OLP) is considered a T cell-mediated chronic inflammatory process activated by an unknown antigen, making basal keratinocytes vulnerable to a cytotoxic cell mediated immune response. The aim of this review is to summarize information on the role and pathways of Epstein–Barr virus (EBV) and immune cells in inducing OLP as an autoimmune lesion. The pathogenesis of OLP is analyzed from immunological aspects of interactions between EBV and oral mucosa. The results of the available studies allow us to assume that EBV can act both as an exogenous and an endogenous antigen in the pathogenesis of OLP. We emphasized the role of antigen-presenting cells (APC), such as dendritic cells (Langerhans cells, LC), in detecting and capturing antigens and modulating the adaptive immune response. Although EBV shows tropism for B cells and epithelial cells, under certain conditions it can infect monocytes, LCs, NK, and T lymphocytes. It means that under some circumstances of the chronic inflammatory process, EBV particles can react as endogenous agents. During the development of the autoimmune process, a decisive role is played by the loss of immune tolerance. Factors like the activity of cytokines, chemokines, and autoantibodies secreted by EBV-positive plasma cells, autoantigens formed due to virus protein mimicry of human proteins, new self-peptides released from damaged tissues, self-reactive B and T cells, dysregulation of LC function, the anti-apoptotic effect of EBV early lytic antigens, and an imbalance between inflammatory and anti-inflammatory immune cells facilitate the development of an autoimmune process.

Epstein-Barr virus dynamics and its prognostic impact on nasopharyngeal cancers in a non-endemic region.

Epstein-Barr virus (EBV) DNA quantification in nasopharyngeal cancer (NPC) is an indicator of the tumour burden, stage and survival. Although EBV dynamics in endemic regions has been extensively studied and reported, the data from non-endemic regions is sparse. This study attempts to investigate the EBV dynamics in NPC patients from a non-endemic region and also to identify the factors impacting the outcomes. This was a prospective observational study conducted at a tertiary care centre in South India and enrolled patients with non-metastatic, biopsy proven NPC, who were suitable for radical chemo-radiotherapy with or without induction chemotherapy. Two blood samples, one prior to initiation of any anticancer treatment, and second at 6 weeks post treatment, were collected to quantify EBV DNA using real-time quantitative polymerase chain reaction. Antibodies against EBV viral capsid antigen (EBV VCA IgM), EBV Early Antigen (EBV EA IgG) and EBV Nuclear Antigen (EBV EBNA IgG) were also measured in the sample. The impact of EBV dynamics on the outcomes was then analysed. The study included a total of 35 patients. Thirty-three had identifiable EBV DNA (94.3%) and a histological diagnosis of non-keratinising undifferentiated type of squamous cell carcinoma. There was no correlation between the EBV DNA and anti-EBV antibodies. There was a significant association between composite stage and pre-treatment DNA titre (p = 0.030). The mean EBV DNA titre was lower for patients with no clinically demonstrable disease at last follow-up and the reduction in EBV DNA titres was significant (p = 0.020) for those patients who remained disease free. Plasma EBV DNA is an accurate and reliable biomarker for NPC for WHO type 2 and 3 tumours even in non-endemic regions.

Association between Epstein-Barr virus serological reactivation and psychological distress: a cross-sectional study of Japanese community-dwelling older adults

Reactivation of Epstein-Barr virus (EBV) is associated with the etiopathogenesis of a broad spectrum of diseases. This study aimed to investigate the association between psychological distress and EBV serological reactivation among community-dwelling older people and assess the role of sex differences in this association. This population-based cross-sectional survey was conducted among individuals who underwent annual health checkups (N = 2,821; median age 72.4 years). EBV serological reactivation was defined as elevation of EBV early antigen immunoglobulin G titers, and psychological distress was defined as Kessler 6 scores ≥5. Multivariable logistic regression analysis was performed to calculate odds ratios (OR) and 95% confidence intervals (CI) for EBV serological reactivation and psychological distress. EBV serological reactivation and psychological distress were detected in 16.4% and 8.7% of participants, respectively. Women accounted for 71% (328/463) of those with EBV serological reactivation. Multivariable logistic regression analysis showed psychological distress was not significantly associated with EBV serological reactivation among all participants (OR 1.31, 95% CI: 0.95, 1.82; P = 0.102). A sex-stratified multivariable analysis showed a positive association among women (OR 1.45, 95% CI: 1.01, 2.08; P = 0.043), but no association among men. EBV serological reactivation was independently associated with psychological distress in community-dwelling older women. The sex difference in our results warrants further investigation to clarify the physiological mechanisms underlying the association.

Cardiac glycosides from the roots of Streblus asper Lour. with activity against Epstein-Barr virus lytic replication

Cardiac glycosides (CGs) show potential broad-spectrum antiviral activity by targeting cellular host proteins. Herein are reported the isolation of five new (1–5) and eight known (7–13) CGs from the roots of Streblus asper Lour. Of these compounds 1 and 7 exhibited inhibitory action against EBV early antigen (EA) expression, with half-maximal effective concentration values (EC50) being less than 60 nM, and they also showed selectivity, with selectivity index (SI) values being 56.80 and 103.17, respectively. Preliminary structure activity relationships indicated that the C-10 substituent, C-5 hydroxy groups, and C-3 sugar unit play essential roles in the mediation of the inhibitory activity of CGs against EBV. Further enzyme experiments demonstrated that these compounds might inhibit ion pump function and thereby change the intracellular signal transduction pathway by binding to Na+/K+-ATPase, as validated by simulated molecular docking. This study is the first report that CGs can effectively limit EBV lytic replication, and the observations made in this study may be of value for lead compound development.

Human herpesvirus infections and circulating microvesicles expressing galectin-3 binding protein in patients with systemic lupus erythematosus.

Circulating microvesicles (MVs) expressing the type 1 interferon-inducible protein galectin-3 binding protein (G3BP) are potentially major sources of autoantigens in systemic lupus erythematosus (SLE). In this study, we explore if plasma concentrations of G3BP-expressing MVs correlate with signs of various active human herpesvirus (HHV) infections in SLE patients, suggesting a virus-induced mechanism for the generation of these vesicles. In 49 SLE patients, the plasma levels of immunoglobulin G (IgG) against cytomegalovirus (CMV) pp52, Epstein-Barr virus (EBV) early antigen diffuse (EA/D), and HHV6 p41 were measured by ELISAs and used as humoral markers of ongoing/recently active viral infection. MVs in platelet-poor plasma were quantified and characterised by flow cytometry, with regard to the binding of Annexin V (AnxV) and the expression of G3BP. Spearman's rho and the Wilcoxon rank-sum test were applied for associative evaluation of virus serology with MV subsets, and clinical and demographic data. The CMV pp52-directed antibodies correlated positively with the high G3BP-expressing MVs; either low (rho=0.4, p-value=0.005) or high (rho=0.37, p-value=0.01) in AnxV-expression. Furthermore, these MV subsets were higher in individuals with high and low IgG levels against CMV pp52 and EBV EA/D, respectively, relative to subjects with low and high IgG levels against these HHV antigens. Importantly, none of the associations were explained by immunosuppressants or antimalarials. Ongoing/recently active CMV infection is associated with circulating MVs expressing G3BP in SLE patients, supporting a link between specific viral infections and potentially pathogenic MVs in SLE.

Acute Epstein‐Barr virus associated haemophagocytosis in an Asian female: What is the diagnosis?

Acute <scp>Epstein‐Barr</scp> virus associated haemophagocytosis in an Asian female: What is the diagnosis?

Investigation of Long COVID Prevalence and Its Relationship to Epstein-Barr Virus Reactivation

Coronavirus disease 2019 (COVID-19) patients sometimes experience long-term symptoms following resolution of acute disease, including fatigue, brain fog, and rashes. Collectively these have become known as long COVID. Our aim was to first determine long COVID prevalence in 185 randomly surveyed COVID-19 patients and, subsequently, to determine if there was an association between occurrence of long COVID symptoms and reactivation of Epstein–Barr virus (EBV) in 68 COVID-19 patients recruited from those surveyed. We found the prevalence of long COVID symptoms to be 30.3% (56/185), which included 4 initially asymptomatic COVID-19 patients who later developed long COVID symptoms. Next, we found that 66.7% (20/30) of long COVID subjects versus 10% (2/20) of control subjects in our primary study group were positive for EBV reactivation based on positive titers for EBV early antigen-diffuse (EA-D) IgG or EBV viral capsid antigen (VCA) IgM. The difference was significant (p < 0.001, Fisher’s exact test). A similar ratio was observed in a secondary group of 18 subjects 21–90 days after testing positive for COVID-19, indicating reactivation may occur soon after or concurrently with COVID-19 infection. These findings suggest that many long COVID symptoms may not be a direct result of the SARS-CoV-2 virus but may be the result of COVID-19 inflammation-induced EBV reactivation.

Serologic markers of Epstein-Barr virus reactivation are associated with increased disease activity, inflammation, and interferon pathway activation in patients with systemic lupus erythematosus

SLE is a clinically heterogeneous disease characterized by an unpredictable relapsing-remitting disease course. Although the etiology and mechanisms of SLE flares remain elusive, Epstein-Barr virus (EBV) reactivation is implicated in SLE pathogenesis. This study examined the relationships between serological measures of EBV reactivation, disease activity, and interferon (IFN)-associated immune pathways in SLE patients. Sera from adult SLE patients (n = 175) and matched unaffected controls (n = 47) were collected and tested for antibodies against EBV-viral capsid antigen (EBV-VCA; IgG and IgA), EBV-early antigen (EBV-EA; IgG), cytomegalovirus (CMV; IgG), and herpes simplex virus (HSV-1; IgG). Serological evidence of EBV reactivation was more common in SLE patients compared to controls as demonstrated by seropositivity to EBV-EA IgG (39% vs 13%; p = 0.0011) and EBV-VCA IgA (37% vs 17%; p = 0.018). EBV-VCA, CMV1, and HSV-1 IgG seropositivity rates did not differ between SLE patients and controls. Furthermore, concentrations of EBV-VCA (IgG and IgA) and EBV-EA (IgG) were higher in SLE patients. SLE patients with high disease activity had increased concentrations of EBV-VCA IgA (mean ISR 1.34 vs. 0.97; p = 0.041) and EBV-EA IgG levels (mean ISR 1.38 vs. 0.90; p = 0.007) compared with those with lower disease activity. EBV reactivation was associated with enhanced levels of the IFN-associated molecule IP-10 (p < 0.001) and the soluble mediators BLyS (p < 0.001) and IL-10 (p = 0.0011). In addition, EBV-EA IgG responses were enriched in two previously defined patient clusters with robust expression of IFN and inflammatory or lymphoid and monocyte responses. Patients in these clusters were also more likely to have major organ involvement, such as renal disease. This study supports a possible role for EBV reactivation in SLE disease activity.

An Absence of Epstein-Barr Virus Reactivation and Associations with Disease Activity in People with Multiple Sclerosis Undergoing Therapeutic Hookworm Vaccination.

Background: Epstein–Barr virus (EBV) infection is strongly associated with multiple sclerosis (MS). Helminth infection can downregulate antiviral immune responses, potentially protecting against MS, but with a theoretical risk for reactivating latent EBV infection. Objective: To investigate parameters of EBV infection and their relationship with disease activity in people with MS (PwMS) therapeutically vaccinated with Necator americanus (hookworm). Methods: Sequential serum samples from 51 PwMS; 26 therapeutically infected (25 larvae) with N. americanus and 25 controls were tested for EBV virus capsid antigen (VCA) IgG and IgM, EBV nuclear antigen-1 (EBNA-1) IgG, and EBV early antigen (EA) IgG. Disease activity was assessed by periodic MRI. Significance was set at p < 0.05. Results: All PwMS were EBV VCA IgG and EBNA-1 IgG positive, and 35.2% were EBV EA IgG positive. EBV antibody levels were generally stable, and EBV reactivation in PwMS was not demonstrated by significant increases in IgG titre over 12 months. Disease activity was most frequent in PwMS possessing high levels of EBV VCA IgG (>600 units/mL) or EBNA-1 IgG (>150 units/mL); however, there was no association with hookworm treatment. Interpretation: Therapeutic hookworm vaccination was not associated with EBV reactivation. Multiple sclerosis disease activity was associated with high levels of EBV VCA IgG or EBNA-1 IgG.

A Randomized Controlled Trial on Evaluation of Plasma Epstein-Barr Virus Biomarker for Early Diagnosis in Patients With Nasopharyngeal Carcinoma.

Early diagnosis of nasopharyngeal carcinoma (NPC) remains a major problem in Southern China. Epstein-Barr virus (EBV) biomarkers have been widely used in NPC screening. This study aims to evaluate the early diagnostic performances of individual EBV biomarkers in NPC. The levels of EBV biomarkers-IgA antibodies against EBV nuclear antigen 1 (EBNA1-IgA), EBV capsid antigen (VCA-IgA), EBV early antigen (EA-IgA), EBV BZLF1 transcription activator protein (Zta-IgA) and IgG antibodies against EBV BRLF1 transcription activator protein (Rta-IgG)-from 106 NPC patients (stage I and II) and 150 normal subjects were measured. VCA-IgA and EA-IgA were detected by immunofluorescence assay (IFA), EBNA1-IgA, Rta-IgG and Zta-IgA were measure by enzyme-linked immunosorbent assay (ELISA), and EBV DNA was detected by qPCR. Statistical analyses of a single index were conducted to evaluate the significance of NPC early diagnosis and TNM classification. The level of EBNA1-IgA, EBV DNA, VCA-IgA, EA-IgA, Rta-IgG and Zta-IgA in early-stage NPC was significantly higher than in healthy controls (all P < 0.001). EBNA1-IgA yielded the biggest area under the curve (AUC) of 0.962 in distinguishing early-stage NPC patients from the normal subjects, with a sensitivity of 91.5% and a specificity of 98.7%. However, EBV biomarker levels were not associated with tumor size (all P > 0.050), whereas four biomarker levels (EBNA1-IgA, EBV DNA, VCA-IgA, EA-IgA) were related to lymph node metastasis (N0 and N1-2), among which EBNA1-IgA and EBV DNA showed good performance. Finally, high correlation was found between VCA-IgA and EA-IgA (r > 0.800). A single EBNA1-IgA exhibits excellent discrimination performance in early diagnosis of NPC and could become a promising marker for NPC screening.

Epstein–Barr virus, cytomegalovirus and BK polyomavirus burden in juvenile systemic lupus erythematosus: correlation with clinical and laboratory indices of disease activity

Clinical and laboratory investigations have revealed that Epstein-Barr virus (EBV) is involved in altered immunological response of systemic lupus erythematosus (SLE). Higher seroprevalence rates of anti-EBV antibodies and increased viral load are demonstrated in adult SLE patients. The prevalence of BK polyomavirus (BKV) reactivation is also suggested to be higher in SLE. Herein, we aimed to evaluate the immune response of children with SLE to EBV antigens in addition to EBV and BKV DNA. We also tried to evaluate whether these serological results differ from another connective tissue disease - juvenile systemic sclerosis (jSS) - and healthy individuals. Serum levels of EBV early antigen diffuse (EA-D) IgG, EBV nuclear antigen-1 IgG, EBV viral capsid antigen (VCA), cytomegalovirus (CMV) IgG, EBV DNA, CMV DNA and urinary BKV DNA were evaluated in healthy controls and in patients with a diagnosis of juvenile SLE (jSLE) and jSS. A total of 70 jSLE patients, 14 jSS patients and 44 sex-matched healthy individuals were involved in the study. EBV VCA was positive in 84.2% of jSLE patients, 85.7% of jSS patients and 36.3% of healthy controls. EBV EA-D IgG positivity was significantly higher in jSLE patients compared to jSS patients and healthy controls (20% vs. 7.1% and 0%, p = 0.005). EBV VCA positivity was associated with malar rash and immunological disorder, but there was no statistical significance in other antibody positivity in terms of clinical and haemogram findings and autoantibody positivity. CMV DNA positivity was present in only 2.8% of jSLE patients. None of the jSS patients or the healthy controls had CMV DNA positivity. EBV DNA and BKV DNA were also negative in all three groups. The results of our study assume a relationship between SLE and EBV, but we could not demonstrate an association between CMV and BKV. The negative DNA results in contrast to serological positivity can be interpreted as an altered and impaired immune system and increased viral susceptibility. These results suggest that EBV contributes to disease continuity, even if it does not directly cause development.

Biological Activities of Flavonoids from the Wood Extract of Artocarpus heterophyllus L. (Jackfruit)

Background: Artocarpus heterophyllus L. (Jackfruit) has been used traditionally as treatment for inflammation and cancer. The aim of this study was to isolate compounds from A. heterophyllus wood extract and evaluate their biological activities such as anti-tumor promoting effect on Epstein-Barr virus early antigen induction, melanogenesis inhibitory activity on the B16 mouse melanoma 4A5 cell line and cytotoxic activity against three human cancer cell lines (HL60, A549, SK-BR-3). Methods: A. heterophyllus wood was extracted with n-hexane and methanol. The ethyl acetate soluble- fraction separated from the methanol extract was separated and purified with column chromatography to isolate compounds. The structures of isolated compounds were elucidated with spectroscopic methods. These compounds were evaluated for their biological activities. Results: Thirteen known compounds including four prenylflavonoids were isolated from the wood extracts. Nine flavonoids (2, 3, 5-11) exhibited potent anti-tumor promoting activity with IC50 values of 259-296 molar ratio / 32 pmol TPA. Two flavonoids, Norartocarpetin (6) at concentration of 30 μM and cyanomaclurin (11) at the concentration of 100 μM showed melanin content value of 47.6 % and 80.1 %, respectively. Two prenylflavonoids, cudraflavone B (2) and artocarpin (5), showed cytotoxicity against the human cancer cell lines tested. Cudraflavone B (2) showed cytotoxicity against all three human cancer cell lines whereas artocarpin (5) only exhibited cytotoxicity against two out three cell lines testes. The IC50 values were comparable to or better than cisplatin. Conclusion: From the view point of structure activity relationships of the flavonoids isolated, side chains such as prenyl and 3-methyl-1-butenyl moiety were key for their potent biological activities.

The role of toll-like receptor 9 (TLR9) in Epstein-Barr virus-associated gastric cancer

Abstract Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is the most common malignancy caused by EBV infection. Toll-like receptors (TLRs) as major components of innate immune system are crucial in the development of inflammatory processes and carcinogenesis. The aim of our study was to evaluate tissue and serum level of TLR9 in EBV-positive and EBV-negative gastric cancer patients. The study involved 30 EBV(+) and 30 EBV(-) patients. EBV DNA was detected in fresh frozen tumor tissue. In serum samples TLR9 level, transforming growth factor β (TGFβ), interleukin 10 (IL-10) and antibodies against EBV were detected using ELISA tests. TLR9 level was also measured in homogenate of tumour tissue. TLR9 level was statistically lower in EBV(+) patients both in serum and tissue, with statistically higher level in tissue than in serum. Lower level of TLR9 was accompanied by higher level of Epstein-Barr virus capsid antigen (EBVCA), Epstein-Barr virus nuclear antigen (EBNA) and early antigen (EA). A lower level of TLR9 was detected in patients with poorly differentiated cancer (G3) and greater lymph nodes involvement (N3-N4). Lower level of TLR9 in patients with EA may point to TLR9 role in reactivation of EBV infection.

Combination of serum matrix metalloproteinase-3 activity and EBV antibodies improves the diagnostic performance of nasopharyngeal carcinoma.

Objective: Nasopharyngeal carcinoma (NPC) is a malignant head and neck tumor that is highly prevalent in Southeast Asia. The two traditional NPC markers VCA-IgA (EBV viral capsid antigen) and EA-IgA (EBV early antigen) are limited in the screening and diagnosis of NPC. The purpose of present study is to evaluate the diagnostic value of matrix metalloproteinase-3 (MMP3) in NPC.Methods: The levels of 23 secretory MMPs in serum samples from 15 healthy controls and 26 NPC patients were detected by Cytokine Antibody Array 2000. Immunohistochemistry, Real-time PCR and western bolt were used to detect MMP3 mRNA and protein levels in NPC tissues and cell lines. The serum protein levels of MMP3 were further measured by ELISA in healthy control individuals (n = 200) and NPC patients (n = 206).Results: MMP3 can be expressed and secreted by both NPC and fibroblast cell lines, suggesting that the higher expression of MMP3 protein in both tumor nests and stromal of NPC tissues may be the source of circulating MMP3 in NPC patients. Furthermore, we found out both MMP3 concentration and enzymatic activity were significantly increased in the NPC group (n = 206) than the healthy control group (n = 200) (P < 0.001). However, serum MMP3 enzymatic activity, but not MMP3 concentration, was significantly associated with the progression of NPC. In addition, serum MMP3 activity was more valuable in diagnosis of NPC than its concentration (0.86 vs. 0.78, AUC), and MMP3 activity can improve the diagnosis of NPC by combining with EBV-infection biomarkers VCA-IgA and EA-IgA with a sensitivity of 91.5% and a specificity of 92.3%.Conclusions: This study suggested the combination of MMP3 activity and EBV antibodies may be a useful biomarker for screening and diagnosis of NPC.

Comparison of TGF-β, IL-10 levels and LMP-1 in gastric and oropharyngeal carcinoma associated with EBV infection

Abstract Increasing interest has been focused on the Epstein-Barr Virus (EBV)-associated cancers, including oropharyngeal cancer (OPC) and gastric cancer (GC). Different cytokines, growth factors and proteins take part in oncogenesis. The aim of our study was to generate a comparison of interleukin 10 (IL-10) and transforming growth factor β (TGF-β) levels, as well as latent membrane protein (LMP-1), Epstein-Barr virus capsid antigen (EBVCA), Epstein-Barr virus nuclear antigen (EBNA) and early antigen (EA) frequency in the serum of patients with GC and OPC. The study involved 50 patients with diagnosed GC and 50 patients with OPC. All studied patients were EBV positive. Fresh-frozen tumor tissue fragments were tested using nested PCR assay for EBV DNA detection. Sera from all individuals were investigated using ELISA tests to detect the presence of EBVCA IgG, EBNA IgG, EA IgG, as well as to determine the levels of IL-10 and TGF-β. The obtained results were subjected to statistical analysis. In patients with GC, the levels of TGF-β and IL-10 were significantly higher than in OPC patients. However, the frequency and level of EBVCA, EBNA and EA in patients with OPC and GC were not significantly different. In contrast, TGF-β and IL-10 levels were significantly higher in EBVaGC, as compared to OPC, suggesting their role in gastric carcinogenesis. The differences in frequency of LMP-1 detection in patients with OPC and GC may suggest different mechanism of oncogenesis. Further studies are required to clarify the role of Epstein-Barr virus in cancer development.

Epstein-Barr Virus Antibody Titers Are Not Associated with Gastric Cancer Risk in East Asia.

Epstein-Barr virus (EBV)-positive gastric cancers represent a distinct subtype of gastric cancers and account for nearly 10% of the gastric cancer burden, yet risk detection strategies for this cancer subtype are lacking. We conducted a nested case-control study where we assayed 4 EBV antigens [viral capsid antigen (VCA), early antigen (EA), Epstein-Barr nuclear antigen (EBNA), and BZLF1-encoded replication activator protein (ZEBRA)] in either sera or plasma from 1447 gastric cancer cases and 1797 controls obtained from seven prospective cohorts representing individuals from the high gastric cancer-risk countries of China, Japan, and Korea. The prevalence of EBV sero-positivity was universal with the exception of one sero-negative individual, and the highest titers of the EBV antigens VCA (OR 0.95, 95% CI 0.78-1.17), EBNA (OR 0.88, 95% CI 0.72-1.08), EA (OR 0.97, 95% CI 0.79-1.19), and ZEBRA (OR 0.87, 95% CI 0.71-1.07) were not associated with risk of incident gastric cancer. When we stratified these data by H. pylori status, there was no change in the association. Multiplex serology of the aforementioned EBV antigens in serum may not be a suitable biomarker for predicting gastric cancer risk in East Asian populations.

HLA II genes distribution in Epstein–Barr virus-associated nasopharyngeal carcinoma and other tumors of the oral cavity patients in Russia

Background. It has been proved that for the nasopharyngeal carcinoma (NPC) the etiological agent is the Epstein–Barr virus (EBV). Being an ubiquitous infection, EBV, under certain conditions, is able to display its oncogenic potential. Among a wide range of tumors associated with EBV, the NPC occupies a special place because it is characterized by a geographically and ethnically heterogeneous distribution, suggesting that in the pathogenesis of NPC, in addition to EBV, an important role is played by other factors, such as genetic predisposition to this neoplasm. Among known genetic factors influencing the frequency of NPC development, the human leukocyte antigen (HLA) complex occupies an important place, as it plays a central role in the presentation of viral antigens to the immune system. In Russia, the association of HLA alleles with the risk of EBV associated forms of NPC development and with development of other oral cavity tumors (OOCT), not associated with the virus, has not been studied. In the literature there are contradictory information about HLA genes, which determine the predisposition to the emergence of these tumors, and their role in the initiation and formation an immune response to EBV proteins. Objective: to study the distribution of the of DQA1-, DQB1-, DRB1-HLA class II gene variants associated with respectively the risk or resistance to the development of NPC and OOCT and with a high and low level of antibody response to EBV main proteins. A group of healthy persons served as a control. Materials and methods. Blood samples from 62 patients with NPC, 44 patients with OOCT, and as control, 300 healthy individuals, were used in the study. The blood serum samples of NPC and OOCT patients were tested for the presence of immunoglobulin classes G and A antibodies to capsid and early EBV antigens by indirect immunofluorescence. All serum samples of patients and healthy individuals were genotyped on HLA-DQA1, -DQB1 and -DRB1 by the method of multi-primer amplification by sequence-specific primers by real-time polymerase chain reaction. Results. In NPC patients, an increase in the frequency of HLA-DRB1*08 was found when compared with the frequency of a similar allele in healthy individuals (5.6 % vs 1.8 %; odds ratio (OR) 3.2; 95 % confidence interval (CI) 1.1–9.1; p = 0.02), and, on the contrary, a lower HLA-DQB1*0301 frequency was detected (16.1 % vs 25.3 %; p <0.05) than in healthy individuals. The data obtained suggest that the HLA-DRB1*08 gene is associated with an increased sensitivity to NPC. In OOCT patients, HLA-DQB1*0502–4 and HLA-DRB1*16 variants were less common than in healthy individuals (1.1 % vs 6.8 %; p <0.05 and 1.1 % vs 6.7 %; OR 0.16; 95 % CI 0.01–1.08; p <0.05, respectively), suggesting that the HLA-DQB1*0301 gene is associated with resistance to NPC, and HLA-DQB1*0502–4 and HLA-DRB1*16 variants – with resistance to OOCT. It is interesting to note the difference in the frequency of HLA-DRB1*13 between NPC and OOCT patients (17.7 % vs 6.8 %; OR 2.9; 95 % CI 1.1–8.6; p <0.05). One can suggest that this difference is related to the proven involvement of EBV in the NPC development. There were no other differences in the frequencies of class II HLA genes between the groups of NPC and OOCT patients. For the first time in Russia the importance of alleles DQA1, DQB1 and DRB1 of the HLA gene for the NPC and OOCT development, malignant tumors, respectively associated and non-associated with EBV, was studied. The results of the investigation completed together with known literature data allow us to conclude that the above alleles of the HLA class II gene can serve as a factor predisposing to the development of NPC in Russia. Conclusion. However, in order to establish a strict association between a specific HLA haplotype and the NPC and OOCT incidence, the information obtained is insufficient due to the complexity and variability of the genetic control of immune responses controlling the tumor process. A comprehensive study of this issue using different immune response genes and populations of different ethnic origins will probably help to elucidate the effect of genetic polymorphism on the risk of NPC and OOCT development in Russia.

Cytotoxicity, antitumor-promoting and antioxidant activities of Annona muricata in vitro

Annona muricata or soursop belongs to the family Annonaceae and is a plant enriched with annonaceous acetogenin. This plant had been traditionally used for the treatment of various infectious and inflammatory diseases. This study aimed to determine the cytotoxicity, antitumor-promoting and antioxidant activities of Annona muricata various extracts and its isolated acetogenin, annonacin in vitro. All samples were subjected to the inhibition of Epstein-Barr virus early antigen (EBV-EA) activation assay induced by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) on Raji cells. Prior to EBV-EA assay, [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (MTT) assay was performed to determine the samples’ cytotoxicity against Raji cells. Antioxidant activity was performed via 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), ferric reducing antioxidant potential (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays. Annonacin exhibited remarkable cytotoxic activity with IC50 of 2.9 ± 1.3 μM. Inhibition against EBV-EA activation occurred in the following order: hexane leaf > annonacin > aqueous fruit> dichloromethane leaf > methanol leaf > aqueous leaf > ethanol leaf. Methanol leaf extract demonstrated the highest antioxidant activity in all assays and exhibited the most powerful free radical scavenger (87%). Annonacin consistently displayed the lowest antioxidant activity in all assays performed. The findings reported for the first time the antitumor-promoting effects of various extracts of Annona muricata and annonacin in vitro, thus validating their promising anticancer potential.

39.2 VIRAL EXPOSURES AND SCHIZOPHRENIA

BackgroundEpidemiological, immunological, and microbiological studies indicate that infections with members of the family Herpesviridae may be associated with schizophrenia and with cognitive impairment. Herpesviruses are enveloped, double-stranded DNA viruses which are widely prevalent and which are capable of causing persistent infections. The most highly replicated association is that between the alpha herpesvirus Herpes Simplex Virus Type 1 (HSV-1) and cognitive impairment in schizophrenia. Acute HSV-1 infection results in oral lesions which usually resolve spontaneously. However, latency can occur in nerve root ganglia leading to cycles of reactivation in later life. Other herepsviruses may also be associated with schizophrenia. Epstein Barr virus (EBV) is a gamma herpesvirus usually acquired in childhood or adolescence. Acute EBV infection is often associated with fever and adenopathy leading to a vigorous immune response and the suppression of viral replication. However, latency can occur with long term consequences to the infected individual.MethodsWe examined the association between HSV-1 seropositivity and cognitive functioning in 828 individuals with schizophrenia from the Sheppard Pratt cohort and 573 control individuals. We also studied antibodies to EBV in a recently enrolled subset of the Sheppard Pratt cohort consisting of 397 individuals with schizophrenia and 289 without a psychiatric disorder. Antibodies to HSV-1 and EBV proteins were measured by immunoassay and confirmed by Western blot. Cognitive functioning was measured by the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). Regression models were employed to define the independent association between virus exposure and outcome.ResultsSerological evidence of exposure to HSV-1 was associated with significantly lower levels of cognitive functioning as measured by the RBANS Total score (coefficient =-3.84, 95% CI -5.60, -2.09, p<.0001). The strongest association was in the domain of Immediate Memory (coefficient= -4.95, 95% CI -7.24, -2.66, p<.0001.) There was a smaller but statistical significant relationship between serological evidence of exposure to HSV-1 and RBANS Total score in control individuals. (coefficient =-1.98, 95% CI -3.88, -.094, p=.04).In terms of EBV, we found that individuals with schizophrenia had significantly higher levels of antibodies to the EBV viral capsid antigens (VCA) as compared to controls (coefficient= .57, 95% CI .37- .77, p<1.7 10-8). On the other hand, the level of antibody to the EBV Nuclear Antigen (EBNA) and EBV Early Antigen (EA) did not differ between the groups. Within the schizophrenia group, increased levels of EBV VCA antibodies were associated with older age, female gender, and cigarette smoking but not with clinical or cognitive measures.DiscussionThe mechanism of the association between HSV-1 exposure and cognitive deficits in individuals with schizophrenia may be due to underlying neuroanatomical deficits, immune dysregulation, or gene x environmental interactions. The aberrant immune response to EBV may represent underlying immunopathy or exposure to variant viral strains. Other herpesviruses, such as Cytomegalovirus, Herpes Simplex Virus Type 2 and Human Herpesvirus Type 6 have also been associated with schizophrenia risk or with cognitive impairment in some populations. The prevention and treatment of herpesvirus infections might lead to new therapeutic modalities for schizophrenia.

Cancer Chemopreventive Activity of Xanthones from Calophyllum elatum

In our continuing search for compounds with antitumor-promoting activity, we screened eight xanthones isolated from Calophyllum elatum Bedd. (Guttiferae) by examining their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12- O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Several compounds tested in this study showed inhibitory activity against EBV, without showing any cytotoxicity. Isogarciniaxanthone E (2) showed more potent activity than any of the other compounds tested. Furthermore, isogarciniaxanthone E (2) exhibited a marked inhibitory effect on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test.