Fluctuations of epstein-barr virus serological antibodies and risk for nasopharyngeal carcinoma: a prospective screening study with a 20-year follow-up

Background

The impact of variation of Epstein-Barr virus (EBV) antibody titers before the development of nasopharyngeal carcinoma (NPC) is still unclear. We analyzed the fluctuations of antibodies against EBV before histopathological diagnosis to assess the risk of NPC and aimed to provide a reliable basis for screening in high risk populations.

Methods

This study was based on a population-based screening program in Sihui County in Guangdong Province of China. A total of 18,986 subjects were recruited in 1987 and 1992, respectively. Baseline and repeated serological tests were performed for IgA antibodies against EBV capsid antigen (VCA/IgA) and early antigen (EA/IgA). Follow-up until the end of 2007 was accomplished through linkage with population and health registers. Cox proportional hazards regression model was used to estimate the relative risk of NPC in association with EBV antibodies. Time-dependent receiver operating characteristic curve (ROC) analysis was used to further evaluate the predictive ability.

Results

A total of 125 NPCs occurred during an average of 16.9 years of follow-up. Using baseline information alone or together with repeated measurements, serological levels of VCA/IgA and EA/IgA were significantly associated with increased risks for NPC, with a striking dose-response relationship and most prominent during the first 5 years of follow-up. Considering the fluctuant types of serological titers observed during the first three tests, relative risk was highest among participants with ascending titers of EBV VCA/IgA antibodies with an adjusted hazard ratio (HR) of 21.3 (95% confidence interval [CI] 7.1 to 64.1), and lowest for those with decreasing titers (HR = 1.5, 95% CI 0.2 to 11.4), during the first 5 years of follow-up. Time-dependent ROC analysis showed that VCA/IgA had better predictive performance for NPC incidence than EA/IgA.

Conclusion

Our study documents that elevated EBV antibodies, particularly with ascending titers, are strongly associated with an increased risk for NPC.     

Clinical and immunological effect of intravesical interleukin-2 on superficial bladder cancer

TheBamHI Z EBV replication activator (ZEBRA) protein is involved in the switch from latency to productive cycle of Epstein-Barr virus. A recombinant ZEBRA protein was synthesized and assessed in enzymelinked immunosorbent assay (ELISA) for serum IgG response in nasopharyngeal carcinoma (NPC) patients. In 100 NPC serum samples that were positive for IgA to the EBV viral capsid antigen (VCA), 75% had IgG anti-ZEBRA antibodies. In contrast, only 3/83 (3.6%) serum samples from healthy donors and 2/50 (4%) from other cancers were positive for IgG to ZEBRA. Interestingly, in a selected group of 100 NPC sera negative for IgA to VCA, 25% contained IgG anti-ZEBRA antibodies. This suggests that the ELISA for IgG anti-ZEBRA may also identify earlier cases of NPC not detected by the conventional immunofluorescence test for IgA to VCA.     

用转染EB病毒基因片段的真核细胞检测鼻咽癌病人的抗EB病毒核抗原-1(EBNA-1)抗体

用转染EBV DNA Bam HI K片段后稳定表达EBNA-1的K_4细胞作为靶细胞,检测50份鼻咽癌病人和38份健康对照者血清中的IgG/EBNA-1抗体;阳性率分别为100％和92％。前者的平均几何滴度为89.4,后者为18.3,前者约为后者的5倍。同一批被检血清经SPA吸收去除IgG竞争性抑制后,鼻咽癌病人的IgA/EBNA-1抗体阳性率达78％(GMT 20.9),健康人的IgA/EBNA-1抗体阳性率仅5.3％,效价亦很低(GMT 5.2)。表明IgA/EBNA-1抗体对鼻咽癌是比较特异的,可考虑作为鼻咽癌血清学诊断的指标之一。     

The utility of saliva for the assessment of anti-pneumococcal antibodies: investigation of saliva as a marker of antibody status in serum

Context: Salivary antibodies may act as non-invasive marker of systemic immunity enabling assessment of vaccination and protection against bacterial infections.

Objective: To assess if levels of anti-pneumococcal (Pn) antibodies in saliva reflect concentrations in serum and determine whether saliva can accurately identify protective concentrations in serum.

Methods: IgG, IgA and IgM antibody levels in paired saliva and serum samples were measured against 12 Pn polysaccharide antigens in 72 healthy adults.

Results: Antibody levels in saliva correlated positively with serum across immunoglobulin classes, most strongly for IgA. Individuals who had protective antibody levels in serum demonstrated significantly higher IgG and IgA salivary antibody concentrations/secretion rates. Salivary IgG and IgA Pn antibodies were able to distinguish between those with/without protective levels in serum for the majority of serotypes. Salivary IgM antibodies were not able to differentiate protective status. Median IgG and IgA Pn salivary parameters were able to identify individuals who had protective levels in serum on ≥8/12 serotypes with moderate accuracy: median IgA secretion rates provided the best sensitivity (73%) and specificity (71%).

Conclusions: These findings suggest that IgG and IgA Pn specific antibodies in saliva may be useful surrogate markers of antibody status in serum.     

Epstein-Barr Virus and Human Papillomavirus Infections and Genotype Distribution in Head and Neck Cancers

ObjectiveTo investigate the prevalence, genotypes, and prognostic values of Epstein-Barr virus (EBV) and human papillomavirus (HPV) infections in Japanese patients with different types of head and neck cancer (HNC).ResultsOf the 209 HNC patients, 63 (30.1%) had HPV infection, and HPV-16 was the most common subtype (86.9%). HPV E6/E7 mRNA expression was found in 23 of 60 (38.3%) HPV DNA-positive cases detected. The site of highest prevalence of HPV was the oropharynx (45.9%). Among 146 (69.9%) HNCs in which EBV DNA was identified, 107 (73.3%) and 27 (18.5%) contained types A and B, respectively, and 124 (84.9%) showed the existence of del-LMP-1. However, only 13 (6.2%) HNCs were positive for EBER, 12 (92.3%) of which derived from the nasopharynx. Co-infection of HPV and EBER was found in only 1.0% of HNCs and 10.0% of NPCs. Kaplan-Meier survival analysis showed significantly better disease-specific and overall survival in the HPV DNA+/mRNA+ oropharyngeal squamous cell carcinoma (OPC) patients than in the other OPC patients (P = 0.027 and 0.017, respectively). Multivariate analysis showed that stage T1–3 (P = 0.002) and HPV mRNA-positive status (P = 0.061) independently predicted better disease-specific survival. No significant difference in disease-specific survival was found between the EBER-positive and -negative NPC patients (P = 0.155).ConclusionsOur findings indicate that co-infection with HPV and EBV is rare in HNC. Oropharyngeal SCC with active HPV infection was related to a highly favorable outcome, while EBV status was not prognostic in the NPC cohort.     

Evaluation of serum antibody response to a newly identified B-cell epitope in the minor nucleocapsid protein L2 of human papillomavirus type 16

The aim of this work was to identify B-cell epitopes in the minor nucleocapsid (L2) protein of human papillomavirus (HPV) type 16 and characterization of allied antibody response. Serum samples of 513 individuals (323 women with various degrees of cervical atypia, 150 men and 40 small children) were available for the study. Synthetic peptides overlapping the L2 protein of HPV 16 twice were applied in ELISA for epitope scanning and antibody determination. An HPV 16 L2 derived dodecamer SGYIPANTTIPF (amino acids 391-402) proved to be the major B-cell epitope. Both IgA antipeptide antibody positivity (range 7-28%) and mean IgA antibody levels (range 13.2 EIU to 42.4 EIU, P < 0.05) increased with the degree of cervical atypia, whereas antipeptide IgG antibodies showed an opposite trend. During a 2-years follow-up significantly (P < 0.0005) decreasing IgA antibody levels to the SGYIPANTTIPF peptide were associated with regression of koilocytotic atypia. Analysis of anti-peptide IgA antibodies of 118 women with known HPV type revealed that a majority of positives had HPV 16/18 DNA. It was concluded that antibody response to the newly discovered peptide was partially type- and disease-specific. Our results also suggest an impairment of the IgG but not IgA class antibody response to HPV 16 in patients with persistent cervical HPV infection.     

鼻咽癌患者血清中抗Epstein-Barr病毒早期和晚期膜抗原抗体的检测

对Epstein-Barr(EB)病毒抗原的研究,发现有淋巴细胞确定的膜抗原(Lydma)、早期抗原(EA)、壳抗原(VCA)、核抗原(EBNA)、早期膜抗原(EMA)和晚期膜抗原(LMA)。除了Lydma抗原外,鼻咽癌患者对上述抗原均产生相应的IgG和IgA抗体。因而研究这些抗体,对阐明EB病毒与鼻咽癌的关系及鼻咽癌的早期诊断都十分有价值。     

Regulation of Microtubule Dynamics through Phosphorylation on Stathmin by Epstein-Barr Virus Kinase BGLF4

Stathmin is an important microtubule (MT)-destabilizing protein, and its activity is differently attenuated by phosphorylation at one or more of its four phosphorylatable serine residues (Ser-16, Ser-25, Ser-38, and Ser-63). This phosphorylation of stathmin plays important roles in mitotic spindle formation. We observed increasing levels of phosphorylated stathmin in Epstein-Barr virus (EBV)-harboring lymphoblastoid cell lines (LCLs) and nasopharyngeal carcinoma (NPC) cell lines during the EBV lytic cycle. These suggest that EBV lytic products may be involved in the regulation of stathmin phosphorylation. BGLF4 is an EBV-encoded kinase and has similar kinase activity to cdc2, an important kinase that phosphorylates serine residues 25 and 38 of stathmin during mitosis. Using an siRNA approach, we demonstrated that BGLF4 contributes to the phosphorylation of stathmin in EBV-harboring NPC. Moreover, we confirmed that BGLF4 interacts with and phosphorylates stathmin using an in vitro kinase assay and an in vivo two-dimensional electrophoresis assay. Interestingly, unlike cdc2, BGLF4 was shown to phosphorylate non-proline directed serine residues of stathmin (Ser-16) and it mediated phosphorylation of stathmin predominantly at serines 16, 25, and 38, indicating that BGLF4 can down-regulate the activity of stathmin. Finally, we demonstrated that the pattern of MT organization was changed in BGLF4-expressing cells, possibly through phosphorylation of stathmin. In conclusion, we have shown that a viral Ser/Thr kinase can directly modulate the activity of stathmin and this contributes to alteration of cellular MT dynamics and then may modulate the associated cellular processes.     

Class IgG,IgM and IgA antibodies againstStaphylococcus aureus antigens in human serum and saliva

Using the ELISA method antibodies against the sonicate, teichoic acid (TA) and exoproducts ofStaphylococcus aureus were determined in sera and saliva of healthy individuals. Main serum antibodies against all the antigens used were shown to be class IgG antibodies. However, antigens of the sonicate stimulated significantly even the systemic IgA response. In the saliva class IgA antibodies predominated, but IgG antibody levels against TA and exoproducts approached the level of IgA antibodies. Levels of IgM antibodies against all antigens tested were low in both the serum and saliva which corresponds with the anamnestic type of response. On the basis of these results one may assume that not only IgG, but also IgA antibodies are important in the systemic immunity against staphylococcal infection and in the immunity of mucous membranes; besides IgA, even class IgG antibodies play an important role.     

Assessment of pulmonary antibodies with induced sputum and bronchoalveolar lavage induced by nasal vaccination against Pseudomonas aeruginosa: a clinical phase I/II study

BackgroundVaccination against Pseudomonas aeruginosa is a desirable albeit challenging strategy for prevention of airway infection in patients with cystic fibrosis. We assessed the immunogenicity of a nasal vaccine based on the outer membrane proteins F and I from Pseudomonas aeruginosa in the lower airways in a phase I/II clinical trial.MethodsN = 12 healthy volunteers received 2 nasal vaccinations with an OprF-OprI gel as a primary and a systemic (n = 6) or a nasal booster vaccination (n = 6). Antibodies were assessed in induced sputum (IS), bronchoalveolar lavage (BAL), and in serum.ResultsOprF-OprI-specific IgG and IgA antibodies were found in both BAL and IS at comparable rates, but differed in the predominant isotype. IgA antibodies in IS did not correlate to the respective serum levels. Pulmonary antibodies were detectable in all vaccinees even 1 year after the vaccination. The systemic booster group had higher IgG levels in serum. However, the nasal booster group had the better long-term response with bronchial antibodies of both isotypes.ConclusionThe nasal OprF-OprI-vaccine induces a lasting antibody response at both, systemic and airway mucosal site. IS is a feasible method to non-invasively assess bronchial antibodies. A further optimization of the vaccination schedule is warranted.     

4号染色体短臂微卫星多态性与鼻咽癌相关性的研究

鼻咽癌（NPC）是一种多因素复杂疾病。其发病过程涉及EB病毒慢性感染、环境致癌因素及宿主基因之间的相互作用。在这一过程中,那些宿主基因在EB病毒感染及鼻咽癌的发生发展中起了关键作用仍不清楚。 本研究的目的是发现与鼻咽癌发生发展中两个关键步骤相关的遗传变异,即EB病毒持续性感染鼻咽部上皮细胞和鼻咽癌的形成。我们在广西梧州市及苍梧县鼻咽癌高发区收集汉族鼻咽癌患者350例、EB病毒壳抗原IgA抗体阳性者(IgA/VCA+)288例和EB病毒壳抗原IgA抗体阴性者(IgA/VCA-)346例。对先前鼻咽癌家系研究显示的鼻咽癌易感区4号染色体短臂（4p15.1-q12）进行了微卫星精细扫描,在 18 Mb的范围内选择34个微卫星标记,包括319个等位基因,对其进行基因分型。比较分析NPC 组和IgA/VCA+组等位基因频率结果显示,9个等位基因与鼻咽癌呈相关,其中5个为易感等位基因（OR=1.51-5.36, p=0.01-0.03）,4个为限制性等位基因（OR值为0.3-0.71, p值为0.02-0.045）。比较分析IgA/VCA+组和IgA/VCA-组及比较所有IgA/VCA+者（包括NPC患者）和IgA/VCA-者等位基因频率的结果显示,12个等位基因与EB病毒壳抗原IgA抗体持续存在相关,其中3个在两组比较中均呈显著相关。等位基因 D4S3241-136 (p=0.004, OR=1.9, 95%CI=1.2-3.0) 和D4S3347-213 (p=0.001, OR=1.6, 95%CI=1.2-2.1) 可增加EB病毒 IgA/VCA抗体形成的危险,为易感基因;而等位基因D4S174-202 (p=0.001, OR=0.5, 95%CI=0.3-0.7) 可限制IgA/VCA抗体的形成。 但上述结果经多因素比较校正后,均失去相关性。我们的研究结果不能 确定该区域与鼻咽癌的形成相关,而另一个家系研究的结果也未得出相关的结果,但本研究却提供了进一步发现鼻咽癌相关基因的研究模式。有关4号染色体短臂 与EB病毒慢性持续感染及鼻咽癌的形成仍值得进一步深入研究。     

Detection of HIV-Gag p24-Specific Antibodies in Sera and Saliva of HIV-1-Infected Adults and in Sera of Infants Born to HIV-1-Infected Mothers

Secretory immunoglobulin A (IgA) is known to play an important role in the mucosal defense against a variety of pathogens. Although the role of IgA antibodies during sexual transmission of HIV is not clear, HIV-specific IgA antibodies have been detected in various mucosal secretions of HIV-infected individuals. Using a monoclonal antibody against human IgA, we established an ELISA system to detect anti-HIV p24 IgA antibodies in sera and saliva. We have analyzed the levels of anti-HIV p24 IgG and IgA antibodies in sera and saliva of 107 and 119 adults, respectively, with HIV infection at different clinical stages, and in the sera of 13 infants born to HIV-infected mothers. The level of anti-HIV p24 IgA antibodies was lower in sera and higher in saliva as compared to that of anti-HIV p24 IgG antibodies. Where the percentage of HIV-specific serum antibody-positive cases decreased with disease progression, that of saliva antibody-positive cases increased in AIDS patients. Among the 13 infants born to HIV-infected mothers, 7 infants were HIV-p24-specific serum IgA positive. These sera were negative for anti-HIV p24 secretory IgA, suggesting that some infants develop their own immune responses against HIV infection. Thus, the detection of HIV-specific IgA antibodies, especially in saliva, could be a simple and reliable test for the diagnosis of HIV infection.     

用重组痘苗病毒感染动物细胞表达的Epstein-Barr病毒膜抗原检查IgA/MA抗体

用EB病毒膜抗原基因重组的痘苗病毒感染动物细胞,其细胞表面可表达EB病毒膜抗原,以此膜抗原作为诊断抗原检测血清中IgA/MA抗体,明显优于常用的B95-8细胞表面膜抗原,从而为研究人群血清抗体反应与鼻咽癌的关系,为鼻咽癌的诊断和普查开辟了新的途径。     

Normal human sera contain antibodies directed at Fab of IgA

Serum samples from 26 normal volunteers were evaluated by isotype-specific ELISA for the presence of IgG and IgM antibodies directed at IgA. Although there were wide variations in antibody levels, anti-IgA antibodies of both isotypes were found in all individuals tested. The anti-IgA activity was detected against a variety of polymeric and monomeric IgA1 and IgA2 myeloma proteins containing both kappa and lambda light chains. By using Fab and Fc fragments generated by incubation of an IgA1 myeloma protein with IgA1 protease, it was shown that the anti-IgA activity was specific for the Fab portion of the IgA molecule. It was also demonstrated that the serum of two individuals contained both IgG and IgM activity directed at autologous affinity-purified IgA. IgM antibody levels against both whole IgA and Fab of IgA were significantly higher than IgG antibody levels. Cells producing anti-IgA antibodies of both isotypes were detected in lipopolysaccharide-stimulated human spleen.     

测定Epstein-Barr病毒IgA/EA抗体的改进方法

用免疫酶法(IE)检查鼻咽癌(NPC)病人血清中IgA/EA抗体,阳性率为73％,几何平均滴度为25,将血清用马抗人IgG血清或葡萄球菌菌体A蛋白(SPA)处理后,以除去竞争性IgG类抗体后,阳性率可增高至92％,几何平均滴度提高到89,有15例NPC病人血清,经马抗人IgG血清处理前IgA/EA抗体为阴性,处理后均呈阳性反应。     

Rheumatoid arthritis–associated autoantibodies in non–rheumatoid arthritis patients with mucosal inflammation: a case–control study

IntroductionRheumatoid arthritis–associated autoantibodies (RA-AAB) can be present in serum years before clinical onset of rheumatoid arthritis (RA). It has been hypothesized that initiation of RA-AAB generation occurs at inflamed mucosal surfaces, such as in the oral cavity or lungs. The aim of this study was to assess systemic presence of RA-AAB in patients without RA who had oral or lung mucosal inflammation.MethodsThe presence of RA-AAB (immunoglobulin A [IgA] and IgG anti-cyclic citrullinated peptide 2 antibodies (anti-CCP2), IgM and IgA rheumatoid factor (RF), IgG anti-carbamylated protein antibodies and IgG and IgA anti-citrullinated peptide antibodies against fibrinogen, vimentin and enolase) were determined in sera of non-RA patients with periodontitis (PD, n = 114), bronchiectasis (BR, n = 80) or cystic fibrosis (CF, n = 41). Serum RA-AAB levels were compared with those of periodontally healthy controls (n = 36). Patients with established RA (n = 86) served as a reference group. Association of the diseases with RA-AAB seropositivity was assessed with a logistic regression model, adjusted for age, sex and smoking.ResultsLogistic regression analysis revealed that IgG anti-CCP seropositivity was associated with BR and RA, whereas the association with PD was borderline significant. IgA anti-CCP seropositivity was associated with CF and RA. IgM RF seropositivity was associated with RA, whereas the association with BR was borderline significant. IgA RF seropositivity was associated with CF and RA. Apart from an influence of smoking on IgA RF in patients with RA, there was no influence of age, sex or smoking on the association of RA-AAB seropositivity with the diseases. Anti-CarP levels were increased only in patients with RA. The same held for IgG reactivity against all investigated citrullinated peptides.ConclusionAlthough overall levels were low, RA-AAB seropositivity was associated with lung mucosal inflammation (BR and CF) and may be associated with oral mucosal inflammation (PD). To further determine whether mucosal inflammation functions as a site for induction of RA-AAB and precedes RA, longitudinal studies are necessary in which RA-AAB of specifically the IgA isotype should be assessed in inflamed mucosal tissues and/or in their inflammatory exudates.     

用PO4细胞为靶细胞检测人血清中Epstein-Barr病毒IgA/MA抗体以诊断鼻咽癌

近年来,常用未经丙酮固定的、由丁酸和巴豆油激活的B95-8细胞或P3HR-1细胞为靶细胞,检测人血清中EB病毒IgA/MA抗体以早期诊断鼻咽癌,效果良好。但由于B95-8细胞含有多种EB病毒抗原,不能用丙酮固定,需多次离心沉淀,在浮悬状态下检测,技术比较复杂。