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Secretory immunoglobulin A in saliva of women with oral and genital HPV infection
European Journal of Obstetrics & Gynecology and Reproductive Biology 124 (2006) 227–231 www.elsevier.com/locate/ejogrb
Secretory immunoglobulin A in saliva of women with oral and genital HPV infection Ana Katherine S. Gonc¸alves a,b, Paulo Giraldo a,b,*, Silvia Barros-Mazon a,b, Marisa Lins Gondo a,b, Rose Luce Amaral a,b, Cla´udia Jacyntho a,b b
a The State University of Campinas, School of Medical Sciences, Sa˜o Paulo, Brazil The Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil
Received 29 January 2005; received in revised form 11 May 2005; accepted 30 June 2005
Abstract Secretory IgA contributes towards the protection of mucosal surfaces against invading microorganisms. Objectives: Quantify secretory IgA titers in the saliva of women with HPV in the oropharynx and/or in the genital area. Subjects and methods: Seventy women with clinical genital HPV lesions and 70 women without HPV infection were tested for oral HPV DNA and the levels of total IgA in their saliva. One millilitre of saliva was collected, centrifuged and stored at 80 8C for the measurement of secretory IgA by nephelometry technique. A pool of oral pharyngeal cells was collected for HPV identification by polymerase chain reaction. Results: Oral HPV PCR was positive in 29 (21%) women (26 women with genital HPV and only 3 women without genital HPV). Titers of secretory IgA were extremely lower in-patients with HPV DNA in the oropharynx when compared to HPV negative women ( p < 0.0001). Genital HPV and smoking were also associated to low levels of total sIgA in saliva ( p < 0.01). After multivariable analyses only the presence of HPV in the oral cavity and/or in genital area, but not smoking, was related to low levels of total secretory IgA. Conclusion: Women with low levels of total secretory IgA could be more susceptible to having their oral mucosa colonized by HPV. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Human Papillomavirus; Sexually transmitted disease; Secretory IgA; Mucousa immunity; Vaginal and oral infection
1. Introduction Human Papillomavirus (HPV) is today one of the most prevalent sexually transmitted viruses worldwide [1]. Its specificity by the skin and mucous epithelium lead to condylomas, papillomas and malignant neoplasias [1–3]. Persistent HPV infection is a necessary factor but not sufficient itself to explain the development of cervical cancer. For the majority of the adult population, it is asymptomatic and transient, presenting no clinical or sub clinical manifestations. Despite the high prevalence of HPV infection only a small percentage of individuals develop malignant neoplasias. The presence of the virus in the tissue is not enough to * Corresponding author. Present address: Rua Dom Francisco de Campos Barreto 145, Campinas, 13092-160 Sa˜o Paulo, Brazil. Tel.: +55 19 32942292; fax: +55 19 37889302. E-mail address: [email protected] (P. Giraldo).
explain the transformation of an infectious process into a neoplasia, even when the infection is due to high risk types such as HPV 16 or HPV 18 [3,4]. In addition to the virus, other factors are necessary to transform the infection into a pre-neoplasic lesion. It is interesting to observe that the clinical and sub clinical manifestations of HPV occur much more frequently in the genital area than in the oropharynx. In 1996, Giraldo et al. noted by means of cytological smears of the oral cavity, that there was strong cytological evidence of HPV infection in 6% of patients with genital HPV [5]. Terai et al. [6] using PCR, observed a high prevalence (56.7%) of HPV in the normal oral cavities of adults from both sexes. An individual’s immunological status seems to be the main determining factor for the progression and recurrence of HPV. Secretory immunoglobulin A (sIgA), known to be important in the control of various diseases on mucosal surfaces, may have a role in preventing the development of HPV infection in the oral mucosa. This immunoglobulin is
0301-2115/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2005.06.028
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found mainly in the gastrointestinal tract and in the mucosal secretions such as saliva, breast milk, traquealbronchal and genitourinary secretions [7,8]. The sIgA prevents microbial adherence to cells and offers protection against intracellular pathogens [9]. In the oral cavity, sIgA predominates in the external secretions and in the interior of the salivary glands. The objective of this study was to quantify the levels of the total sIgA in saliva of women with or without HPV in the oral and/or genital mucosa. Our hypothesis is that women infected by HPV have reduced levels of sIgAs in their saliva, and therefore, that measurement of this immunoglobulin could be a prognostic marker for the evolution of HPV disease.
2. Subjects and methods Seventy sexually active female patients from the outpatient clinic of genital infections at the State University of Campinas, SP, Brazil who presented a clinical HPV genital lesion, confirmed by a histopathological examination from August 2001 to August 2002, were invited to participate in the study. The control group was composed by the same number of sexually active women without any macroscopic or sub clinical HPV lesions. Routine pap smear and colposcopic examinations were performed on all participants to exclude the possibility of sub clinical HPV lesions. Women who were pregnant, menopausal, using immunosuppressive medications or antibiotics, presenting oral infections and/or inflammations or degenerative diseases were excluded. All women were tested for HPV DNA in the oral mucosa by polymerase chain reaction. The levels of sIgA in the saliva were quantitated, quantified by nephelometry technique. This study was approved by the Medical Ethics Committee at the School of Medical Science; the State University of Campinas, Brazil and informed consent was obtained before enrolling the women in the study. 2.1. Sampling of the material from the oral cavity Mucosal cells were collected by gently swabbing the hard palate and buccal cavity with sterile Dacron swabs. The material was placed in 1 ml phosphate-buffered saline (PBS) and frozen at 80 8C. One millilitre of saliva was aspirated from the mouth of each patient using a 5 ml syringe and placed in an Eppendorf tube. The material was immediately centrifuged (centrifuge—5415C Eppendorf, 600 g) for 12 min and frozen at 80 8C for the posterior measurement of sIgA. 2.2. HPV DNA amplification by PCR and sIgA measurement The frozen cellular material was thawed and processed; DNA was extracted and analyzed by PCR for HPV detection, using consensus MY09 and MY11 primers that
flank the L1 region of the HPV genome [12]. Quality control for DNA integrity and the reaction process was performed by amplification of the human m-globulin gene, using RS42 and KM29 primers. Positive and negative control samples were tested in parallel to the test samples [12]. The non-diluted saliva samples were submitted to total sIgA quantification by nephelometry technique with human IgA reagent and BN 2 nephelometry (DadeBehring, Marburg, Germany). All the samples were tested in duplicate and the medium value calculated. The device was calibrated to reach sensibility levels of 1.5 mg/dl. The results were initially set up by univariate analysis by means of frequency distributions of the variables. Comparative analysis was performed to measure the association between oral PHV, genital HPV and levels of sIgA. For continuous asymmetric distribution, variables such as the levels of total sIgA in the study groups were established and compared using the Mann–Whitney, Dunn and Kruskal– Wallis non-parametric tests [11]. The magnitude of these associations was expressed by odds ratio (CI 95%). A multivariate logistic regression was performed in order to test the association between each variable and low levels of sIgA and to isolate the influence of each variable. The low and high levels of sIgA was determined by a cut-off value of 5.8 mg/dl based on the median confidence interval (95%) of study cases and controls.
3. Results Oral HPV PCR was positive in 29 (21%) women (26 women with genital HPV and only 3 women without genital HPV. The HPV positive groups and negative groups presented similar mean age [27.7 (6.5) versus 30.1 (8.2)]. No significant differences were found for white race, number of pregnancies, use of contraceptive methods, Table 1 Demographic and sexual characteristics in 140 women with or without DNA HPV in the oral cavity Characteristics
Age (mean S.D.) White race (%) Primary education (%) Secondary education (%) Smoking (%) Number of cigarettes/day (mean S.D.) Parity (mean S.D.) Oral contraceptive (%) Sexual intercourse/month (mean S.D.) Donor oral sex (%) Receptor oral sex (%)
Oral HPV infection
p-Value
Positive (29)
Negative (111)
27.7 6.5 79.3 69.0 31.0 41.4 15.8 9.5
30.1 8.2 72.1 52.2 47.7 26.1 13.6 8.9
NS NS NS NS NS NS
2.3 1.5 72.4 9.6 5.9
2.5 1.9 65.8 8.1 5.4
NS NS NS
58.6 55.2
53.2 53.1
NS NS
Standard deviation: S.D., Mann–Whitney test, x2-test.
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Table 2 Comparison of total secretory IgA levels in women based on the presence of HPV DNA in oral mucosa or presence of genital HPV lesions or previous history of smoking n
Minimum value (mg/dl)
Medium (mg/dl)
Maximum value (mg/dl)
p-Value
70 70
<1.5 <1.5
4.5 8.4
17.0 28.2
<0.0001
29 111
<1.5 <1.5
3.1 8.0
8.6 28.2
<0.0001
41 99
<1.5 <1.5
4.9 7.8
17.0 28.2
0.0042
Genital HPV lesions Positive Negative Oral DNA HPV Positive Negative History of smoking Positive Negative
Mann–Whitney and Kruskal–Wallis test.
history of smoking, level of schooling, mean number of sexual intercourse/month, frequency of donor oral sex and receptor oral sex (Table 1). Statistical analysis compared the medium, maximum, minimum values for the total sIgA levels, based on the presence of the HPV DNA in the oral cavity, the presence of genital HPV lesions or a history of smoking. The patients with oral HPV DNA, genital HPV lesions and smokers had significantly lower values of sIgA than their respective controls (Table 2). Women, who presented positive results for Oral HPV, genital HPV and history of smoking had even lower levels of total salivary sIgA when compared to women with negative results. In the multivariate logistic regression, there was a strong association between low levels of sIgA and the presence of HPV DNA in the oral cavity (OR = 51.63; CI: 11.4–233.3) and genital HPV lesions (OR = 9.58; CI: 4.11– 9.3), respectively (Table 3).
4. Discussion HPV infection is not strictly limited to the genital organs; it can extend itself to extragenital areas (oral) [1]. Secretory IgA is the primary effector of mucosal immunity, and its role is preponderant over the immunoglobulins activities [12]. During an infection, secretory IgA inhibits the adherence of pathogenic microorganisms to the mucosal cell membrane, in addition to having the ability to interfere with the motility of bacteria, and neutralizing their products. The sIgA neutralizes intracellular viruses, where usually the antibodies offer little protection against intracellular pathogens [9]. Various authors [13–15] have proposed a single dichotomous model (Th1/Th2) for the immune response, to explain the natural history of HPV infections, where cell mediated immunity carries out a single prevalent role and
Table 3 Multivariate logistic regression testing the association of different variables and low levels of total secretory IgA in saliva of women with and without HPV infection Characteristics
Secretory IgA levels Low
Brute OR
OR
High
n
%
n
%
Genital HPV lesion Positive Negative
41 9
82.0 18.0
29 61
32.2 67.8
9.58 (4.11–22.3) Ref.
3.1 (1.0–9.3) Ref.
Oral DNA HPV Positive Negative
27 23
54.0 46.0
2 88
2.2 97.8
51.65 (11.4–233.3) Ref.
60.5 (9.7–376) Ref.
History of smoking Positive Negative
22 28
44.0 56.0
19 71
21.1 78.9
2.94 (1.38–6.24) Ref.
1.2 (0.2–6.8) Ref.
Mean
S.D.
Medium
Mean
S.D.
Medium
Brute OR
OR
Age No. of cigarettes/day N. part.
30.3 7.4 8.3
8.1 10.3 4.6
30 0 8
28.3 2.6 8.5
7.5 6.2 6.0
27 0 8
1.0 (0.9–1.0) 1.07 (1.03–1.1) 0.99 (0.9–1.0)
0.9 (0.9–1.0) 1.0 (1.0–1.1) 0.9 (0.8–1.0)
S.D. = Standard deviation.
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the exclusive presence of pro-inflammatory cytokines (IFNg) determine the evolution of the infections induced by HPV. Contrary to this present way of thinking, it is possible for the protection against HPV to be carried out by means of the transference of ‘‘naive’’ immonoglobulins [16,17]. It was also observed that the parenteral injection of VLPS (particles similar to the virus), induced the protection of neutralized serum antibodies that give protection against infections in experimental models (rats). In these animals, the presence of neutralized antibodies (IgA and IgG) in the mucosa and in the secretions prevents infections caused by HPV [18,19]. It was noted in previous studies that in non-infected women exposed to HIV virus, specific sIgA not only neutralizes the virus in secretions but also induces transcytosis, whereby this antibody neutralizes intracellular virus by blocking assembly of viral antigens [20–23]. Thus, the cellular immune response does not seem to be the only means of host defense against HPV infection. The mucosal humoral defense against HPV infection is not well studied. In the present study, it was seen that subjects having lower levels of total secretory IgA were more susceptible to colonization of the genital and oral mucous by HPV. It should be considered that even though oral and/or genital HPV carriers are systemically immunocompetent, they could have a specific and temporary immunological deficiency for some viral infectious agents (HPV) at a mucous level. It is interesting to note that despite the similarities between the oral and genital mucous, HPV infection is more noticeable in the genital area than in the oral cavity. Swallowing, the brushing of teeth and the production of enzymes can protect mucosa, however, differential sIgA levels could have an important role for the installation and development of HPV. In this study, low levels of total sIgA were consistently related to the presence of HPV DNA in the oral mucosa and to the presence of genital HPV lesions. Multivariate regression, used to identify the relevance of each variable showed that the presence of DNA HPV in the oropharyngeal was consistently related to low levels of total secretory IgA (OR = 60.5; CI 9.7–376). On the other hand, history of smoking lost its initial significance (OR = 1.2; CI 0.2–6.6) as it was basically associated to the presence of genital HPV lesions. Although a history of smoking has been historically associated to neoplasias and pre-neoplasic lesions induced by HPV [24,25], this influence was not present in this study. Considering that the various types of HPV are transmitted by sexual contact, the presence of neutralized antibodies, within the basal cells and in the secretions of the mucous, at the time of infection, can prevent the entrance of the virus and limit its transmission [9,16,19]. In this study, it was verified that the patients who presented with low levels of total secretory IgA were significantly more susceptible to HPV colonization in their oral and genital mucosa.
The authors believe that as the oral mucosa is very similar to the genital mucosa, and that once values and techniques to quantify this important immunoglobulin are established, it would be possible to use the measurement of total sIgA as an interesting parameter for the evaluation of immunity of the mucosa and its susceptibility to certain microorganisms or even to use this measurement as a prognostic factor for the evolution of the disease. It remains to be seen if the variations of the levels of the immunoglobulin would be the cause or consequence of the installation of HPV in this mucosa.
References [1] Wieland U, Pfister H. Papillomavirus in human pathology: epidemiology, pathogenesis and oncogenic role oral. In: Gross GE, Barrasso R, editors. Human Papillomavirus virus infection. Berlin: Wiesbaden; 1997. p. 399–412. [2] Howley PM. Role of the human papillomavirus in human cancer. Cancer Res 1991;51:5019–22. [3] Lorincz AT, Reid R, Jenson AB, Greemberg MD, Lancaster W, Kurman RJ. Human papillomavirus infection of the cervix: relative risk association of 15 common anogenital types. Obstet Gynecol 1992; 79:328–37. [4] Kellokoski J, Syrja¨nen S, Chang F, Yliskoski M, Syrja¨nen K. Southern blot hybridization and PCR in detection of oral human papillomavirus (HPV) infections in women with genital Papillomavirus infections. U Oral Pathol Med 1992;21:459–64. [5] Giraldo PC, Simo˜es JA, Ribeiro-Filho AD, Tambascia JK. Avaliac¸a˜o Citolo´gica da Orofaringe de Mulheres portadoras de HPV Genital. RBGO 1996;18(9):737–42. [6] Terai M, Hashimoto K, Yoda K, Sata T. High prevalence of human papillomavirus in the normal oral cavity of adults. Oral Microbiol Immunol 1999;14:201–5. [7] Abbas AK, Lichtman AH, Pober JS. Cellular and molecular immunology, 4a ed., USA: WB Saunders, 2003. [8] Brandzaeg P, Baekkevold ES, Farstad IM, et al. Regional specialization in the mucosal immune system: what happens in the microcompartiments? Immunol Today 1999;20(3):141–51. [9] Egmon M, Damen CA, Spriel ABV, Vidarsson G, Garderen VE, Winkel JG. IgA and IgA Fc receptor. Trends Immumol 2001; 22(4):205–11. [11] Medronho RA, Carvalho DM, Bloch KV, Luiz RR, Werneck GL. Epidemiologia. Sa˜o Paulo: Atheneu, 2002. [12] Childers NK, Bruce MG, Mcghee JR. Molecular mechanisms of immunoglobulin A defense. Annu Rev Microbiol 1989;43:503–36. [13] Grassegger A, Rollinger-Holzinger I, Zelger BWH, et al. Spontaneous or interferon-g-induced T-cell infiltration, HLA-DR and ICAM-1 expression in genitoanal warts are associated with Th1 or mixed Th1/Th2 cytokine mRNA expression profiles. Arch Dermat Res 1997;289:243–50. [14] Clerici M, Merola M, Ferrario E, et al. Cytokine production patterns in cervical. Intraepithelial neoplasia: association with human papillomavirus infection. J Natl Cancer Inst 1997;89:245–50. [15] Tartour E, Gey A, Sastre-Garau X, Surin IL, Mosseri V, Fridman WH. Prognostic value of intratumoral vaginal interferon gamma messenger RNA expression in invasive cervical carcinomas. J Natl Cancer Inst 1998;90:287–94. [16] Suzich JA, Ghim SJ, Palmer-Hill FJ, et al. Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas. Proc Natl Acad Sci USA 1995; 92(25):11553–7. [17] Breitburd F, Kirnbauer R, Hubbert NL, et al. Immunization with virus like particles from cottontail rabbit Papillomavirus (CRPV) can protect against experimental CRPV infection. J Virol 1995; 69(6):3959–63.
A.K.S. Gonc¸alves et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 124 (2006) 227–231 [18] Thapar MA, Parr EL, Parr MB. Secretory immune responses in mouse vaginal fluid after pelvic, parenteral or vaginal immunization. Immunology 1990;70:121–5. [19] Balmelli C, Roden R, Potts A, Schiller J, De Grandi P, NardelliHaeflinger D. Nasal immunization of mice with Human Papillomavirus Type 16 Virus-like Particles elicits neutralizing antibodies in mucosal secretions. J Virol 1998;72(10):8220–9. [20] Kaul R, Trabattoni D, Bwayo JJ, Arienti D, Zagliani A, Mwangi FM, et al. HIV-1-specific mucosal IgA in a cohort of HIV-1-resistant Kenyan sex workers. AIDS 1999;13(1):23–9. [21] Kaul R, Plummer F, Clerici M, Bomsel M, Lopalco L, Broliden K. Mucosal IgA in exposed, uninfected subjects: evidence for a role in protection against HIV infection. AIDS 2001;15(3):431–2.
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[22] Devito C, Hinkula J, Kaul R, Lopalco L, Bwayo JJ, Plummer F, et al. Mucosal and plasma IgA from HIV-exposed seronegative individuals neutralize a primary HIV-1 isolate. AIDS 2000;14(13): 1917–1920. [23] Devito C, Broliden K, Kaul R, et al. Mucosal and plasma IgA from HIV-1-exposed uninfected individuals inhibit HIV-1 transcytosis across human epithelial cells. J Immunol 2000;165(9): 5170–5176. [24] Brinton LA, Schairer C, Haenszel W, et al. Cigarette smoking and invasive cervical cancer. JAMA 1992;255:528. [25] Gram IT, Austin H, Stalsberg H. Cigarette smoking and the incidence of cervical intraepithelial neoplasia grade III, and cancer of the cervix. Am J Epidemiol 1992;135:341.
Secretory immunoglobulin A in saliva of women with oral and genital HPV infection Ana Katherine S. Gonc¸alves a,b, Paulo Giraldo a,b,*, Silvia Barros-Mazon a,b, Marisa Lins Gondo a,b, Rose Luce Amaral a,b, Cla´udia Jacyntho a,b b
a The State University of Campinas, School of Medical Sciences, Sa˜o Paulo, Brazil The Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil
Received 29 January 2005; received in revised form 11 May 2005; accepted 30 June 2005
Abstract Secretory IgA contributes towards the protection of mucosal surfaces against invading microorganisms. Objectives: Quantify secretory IgA titers in the saliva of women with HPV in the oropharynx and/or in the genital area. Subjects and methods: Seventy women with clinical genital HPV lesions and 70 women without HPV infection were tested for oral HPV DNA and the levels of total IgA in their saliva. One millilitre of saliva was collected, centrifuged and stored at 80 8C for the measurement of secretory IgA by nephelometry technique. A pool of oral pharyngeal cells was collected for HPV identification by polymerase chain reaction. Results: Oral HPV PCR was positive in 29 (21%) women (26 women with genital HPV and only 3 women without genital HPV). Titers of secretory IgA were extremely lower in-patients with HPV DNA in the oropharynx when compared to HPV negative women ( p < 0.0001). Genital HPV and smoking were also associated to low levels of total sIgA in saliva ( p < 0.01). After multivariable analyses only the presence of HPV in the oral cavity and/or in genital area, but not smoking, was related to low levels of total secretory IgA. Conclusion: Women with low levels of total secretory IgA could be more susceptible to having their oral mucosa colonized by HPV. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Human Papillomavirus; Sexually transmitted disease; Secretory IgA; Mucousa immunity; Vaginal and oral infection
1. Introduction Human Papillomavirus (HPV) is today one of the most prevalent sexually transmitted viruses worldwide [1]. Its specificity by the skin and mucous epithelium lead to condylomas, papillomas and malignant neoplasias [1–3]. Persistent HPV infection is a necessary factor but not sufficient itself to explain the development of cervical cancer. For the majority of the adult population, it is asymptomatic and transient, presenting no clinical or sub clinical manifestations. Despite the high prevalence of HPV infection only a small percentage of individuals develop malignant neoplasias. The presence of the virus in the tissue is not enough to * Corresponding author. Present address: Rua Dom Francisco de Campos Barreto 145, Campinas, 13092-160 Sa˜o Paulo, Brazil. Tel.: +55 19 32942292; fax: +55 19 37889302. E-mail address: [email protected] (P. Giraldo).
explain the transformation of an infectious process into a neoplasia, even when the infection is due to high risk types such as HPV 16 or HPV 18 [3,4]. In addition to the virus, other factors are necessary to transform the infection into a pre-neoplasic lesion. It is interesting to observe that the clinical and sub clinical manifestations of HPV occur much more frequently in the genital area than in the oropharynx. In 1996, Giraldo et al. noted by means of cytological smears of the oral cavity, that there was strong cytological evidence of HPV infection in 6% of patients with genital HPV [5]. Terai et al. [6] using PCR, observed a high prevalence (56.7%) of HPV in the normal oral cavities of adults from both sexes. An individual’s immunological status seems to be the main determining factor for the progression and recurrence of HPV. Secretory immunoglobulin A (sIgA), known to be important in the control of various diseases on mucosal surfaces, may have a role in preventing the development of HPV infection in the oral mucosa. This immunoglobulin is
0301-2115/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2005.06.028
228
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found mainly in the gastrointestinal tract and in the mucosal secretions such as saliva, breast milk, traquealbronchal and genitourinary secretions [7,8]. The sIgA prevents microbial adherence to cells and offers protection against intracellular pathogens [9]. In the oral cavity, sIgA predominates in the external secretions and in the interior of the salivary glands. The objective of this study was to quantify the levels of the total sIgA in saliva of women with or without HPV in the oral and/or genital mucosa. Our hypothesis is that women infected by HPV have reduced levels of sIgAs in their saliva, and therefore, that measurement of this immunoglobulin could be a prognostic marker for the evolution of HPV disease.
2. Subjects and methods Seventy sexually active female patients from the outpatient clinic of genital infections at the State University of Campinas, SP, Brazil who presented a clinical HPV genital lesion, confirmed by a histopathological examination from August 2001 to August 2002, were invited to participate in the study. The control group was composed by the same number of sexually active women without any macroscopic or sub clinical HPV lesions. Routine pap smear and colposcopic examinations were performed on all participants to exclude the possibility of sub clinical HPV lesions. Women who were pregnant, menopausal, using immunosuppressive medications or antibiotics, presenting oral infections and/or inflammations or degenerative diseases were excluded. All women were tested for HPV DNA in the oral mucosa by polymerase chain reaction. The levels of sIgA in the saliva were quantitated, quantified by nephelometry technique. This study was approved by the Medical Ethics Committee at the School of Medical Science; the State University of Campinas, Brazil and informed consent was obtained before enrolling the women in the study. 2.1. Sampling of the material from the oral cavity Mucosal cells were collected by gently swabbing the hard palate and buccal cavity with sterile Dacron swabs. The material was placed in 1 ml phosphate-buffered saline (PBS) and frozen at 80 8C. One millilitre of saliva was aspirated from the mouth of each patient using a 5 ml syringe and placed in an Eppendorf tube. The material was immediately centrifuged (centrifuge—5415C Eppendorf, 600 g) for 12 min and frozen at 80 8C for the posterior measurement of sIgA. 2.2. HPV DNA amplification by PCR and sIgA measurement The frozen cellular material was thawed and processed; DNA was extracted and analyzed by PCR for HPV detection, using consensus MY09 and MY11 primers that
flank the L1 region of the HPV genome [12]. Quality control for DNA integrity and the reaction process was performed by amplification of the human m-globulin gene, using RS42 and KM29 primers. Positive and negative control samples were tested in parallel to the test samples [12]. The non-diluted saliva samples were submitted to total sIgA quantification by nephelometry technique with human IgA reagent and BN 2 nephelometry (DadeBehring, Marburg, Germany). All the samples were tested in duplicate and the medium value calculated. The device was calibrated to reach sensibility levels of 1.5 mg/dl. The results were initially set up by univariate analysis by means of frequency distributions of the variables. Comparative analysis was performed to measure the association between oral PHV, genital HPV and levels of sIgA. For continuous asymmetric distribution, variables such as the levels of total sIgA in the study groups were established and compared using the Mann–Whitney, Dunn and Kruskal– Wallis non-parametric tests [11]. The magnitude of these associations was expressed by odds ratio (CI 95%). A multivariate logistic regression was performed in order to test the association between each variable and low levels of sIgA and to isolate the influence of each variable. The low and high levels of sIgA was determined by a cut-off value of 5.8 mg/dl based on the median confidence interval (95%) of study cases and controls.
3. Results Oral HPV PCR was positive in 29 (21%) women (26 women with genital HPV and only 3 women without genital HPV. The HPV positive groups and negative groups presented similar mean age [27.7 (6.5) versus 30.1 (8.2)]. No significant differences were found for white race, number of pregnancies, use of contraceptive methods, Table 1 Demographic and sexual characteristics in 140 women with or without DNA HPV in the oral cavity Characteristics
Age (mean S.D.) White race (%) Primary education (%) Secondary education (%) Smoking (%) Number of cigarettes/day (mean S.D.) Parity (mean S.D.) Oral contraceptive (%) Sexual intercourse/month (mean S.D.) Donor oral sex (%) Receptor oral sex (%)
Oral HPV infection
p-Value
Positive (29)
Negative (111)
27.7 6.5 79.3 69.0 31.0 41.4 15.8 9.5
30.1 8.2 72.1 52.2 47.7 26.1 13.6 8.9
NS NS NS NS NS NS
2.3 1.5 72.4 9.6 5.9
2.5 1.9 65.8 8.1 5.4
NS NS NS
58.6 55.2
53.2 53.1
NS NS
Standard deviation: S.D., Mann–Whitney test, x2-test.
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Table 2 Comparison of total secretory IgA levels in women based on the presence of HPV DNA in oral mucosa or presence of genital HPV lesions or previous history of smoking n
Minimum value (mg/dl)
Medium (mg/dl)
Maximum value (mg/dl)
p-Value
70 70
<1.5 <1.5
4.5 8.4
17.0 28.2
<0.0001
29 111
<1.5 <1.5
3.1 8.0
8.6 28.2
<0.0001
41 99
<1.5 <1.5
4.9 7.8
17.0 28.2
0.0042
Genital HPV lesions Positive Negative Oral DNA HPV Positive Negative History of smoking Positive Negative
Mann–Whitney and Kruskal–Wallis test.
history of smoking, level of schooling, mean number of sexual intercourse/month, frequency of donor oral sex and receptor oral sex (Table 1). Statistical analysis compared the medium, maximum, minimum values for the total sIgA levels, based on the presence of the HPV DNA in the oral cavity, the presence of genital HPV lesions or a history of smoking. The patients with oral HPV DNA, genital HPV lesions and smokers had significantly lower values of sIgA than their respective controls (Table 2). Women, who presented positive results for Oral HPV, genital HPV and history of smoking had even lower levels of total salivary sIgA when compared to women with negative results. In the multivariate logistic regression, there was a strong association between low levels of sIgA and the presence of HPV DNA in the oral cavity (OR = 51.63; CI: 11.4–233.3) and genital HPV lesions (OR = 9.58; CI: 4.11– 9.3), respectively (Table 3).
4. Discussion HPV infection is not strictly limited to the genital organs; it can extend itself to extragenital areas (oral) [1]. Secretory IgA is the primary effector of mucosal immunity, and its role is preponderant over the immunoglobulins activities [12]. During an infection, secretory IgA inhibits the adherence of pathogenic microorganisms to the mucosal cell membrane, in addition to having the ability to interfere with the motility of bacteria, and neutralizing their products. The sIgA neutralizes intracellular viruses, where usually the antibodies offer little protection against intracellular pathogens [9]. Various authors [13–15] have proposed a single dichotomous model (Th1/Th2) for the immune response, to explain the natural history of HPV infections, where cell mediated immunity carries out a single prevalent role and
Table 3 Multivariate logistic regression testing the association of different variables and low levels of total secretory IgA in saliva of women with and without HPV infection Characteristics
Secretory IgA levels Low
Brute OR
OR
High
n
%
n
%
Genital HPV lesion Positive Negative
41 9
82.0 18.0
29 61
32.2 67.8
9.58 (4.11–22.3) Ref.
3.1 (1.0–9.3) Ref.
Oral DNA HPV Positive Negative
27 23
54.0 46.0
2 88
2.2 97.8
51.65 (11.4–233.3) Ref.
60.5 (9.7–376) Ref.
History of smoking Positive Negative
22 28
44.0 56.0
19 71
21.1 78.9
2.94 (1.38–6.24) Ref.
1.2 (0.2–6.8) Ref.
Mean
S.D.
Medium
Mean
S.D.
Medium
Brute OR
OR
Age No. of cigarettes/day N. part.
30.3 7.4 8.3
8.1 10.3 4.6
30 0 8
28.3 2.6 8.5
7.5 6.2 6.0
27 0 8
1.0 (0.9–1.0) 1.07 (1.03–1.1) 0.99 (0.9–1.0)
0.9 (0.9–1.0) 1.0 (1.0–1.1) 0.9 (0.8–1.0)
S.D. = Standard deviation.
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the exclusive presence of pro-inflammatory cytokines (IFNg) determine the evolution of the infections induced by HPV. Contrary to this present way of thinking, it is possible for the protection against HPV to be carried out by means of the transference of ‘‘naive’’ immonoglobulins [16,17]. It was also observed that the parenteral injection of VLPS (particles similar to the virus), induced the protection of neutralized serum antibodies that give protection against infections in experimental models (rats). In these animals, the presence of neutralized antibodies (IgA and IgG) in the mucosa and in the secretions prevents infections caused by HPV [18,19]. It was noted in previous studies that in non-infected women exposed to HIV virus, specific sIgA not only neutralizes the virus in secretions but also induces transcytosis, whereby this antibody neutralizes intracellular virus by blocking assembly of viral antigens [20–23]. Thus, the cellular immune response does not seem to be the only means of host defense against HPV infection. The mucosal humoral defense against HPV infection is not well studied. In the present study, it was seen that subjects having lower levels of total secretory IgA were more susceptible to colonization of the genital and oral mucous by HPV. It should be considered that even though oral and/or genital HPV carriers are systemically immunocompetent, they could have a specific and temporary immunological deficiency for some viral infectious agents (HPV) at a mucous level. It is interesting to note that despite the similarities between the oral and genital mucous, HPV infection is more noticeable in the genital area than in the oral cavity. Swallowing, the brushing of teeth and the production of enzymes can protect mucosa, however, differential sIgA levels could have an important role for the installation and development of HPV. In this study, low levels of total sIgA were consistently related to the presence of HPV DNA in the oral mucosa and to the presence of genital HPV lesions. Multivariate regression, used to identify the relevance of each variable showed that the presence of DNA HPV in the oropharyngeal was consistently related to low levels of total secretory IgA (OR = 60.5; CI 9.7–376). On the other hand, history of smoking lost its initial significance (OR = 1.2; CI 0.2–6.6) as it was basically associated to the presence of genital HPV lesions. Although a history of smoking has been historically associated to neoplasias and pre-neoplasic lesions induced by HPV [24,25], this influence was not present in this study. Considering that the various types of HPV are transmitted by sexual contact, the presence of neutralized antibodies, within the basal cells and in the secretions of the mucous, at the time of infection, can prevent the entrance of the virus and limit its transmission [9,16,19]. In this study, it was verified that the patients who presented with low levels of total secretory IgA were significantly more susceptible to HPV colonization in their oral and genital mucosa.
The authors believe that as the oral mucosa is very similar to the genital mucosa, and that once values and techniques to quantify this important immunoglobulin are established, it would be possible to use the measurement of total sIgA as an interesting parameter for the evaluation of immunity of the mucosa and its susceptibility to certain microorganisms or even to use this measurement as a prognostic factor for the evolution of the disease. It remains to be seen if the variations of the levels of the immunoglobulin would be the cause or consequence of the installation of HPV in this mucosa.
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