Journal of Investigative and Clinical Dentistry (2015), 6, 16–24
ORIGINAL ARTICLE Oral Biosciences
Lactoferrin levels in gingival crevicular fluid and saliva of HIV-infected patients with chronic periodontitis Sonia Maria Soares Ferreira1, Lucio Souza Goncßalves2,3, Sandra R. Torres4, Susie A. Nogueira5 & Timothy F. Meiller6 1 2 3 4 5 6
, Brazil Educational Foundation Jayme de Altavila, Maceio Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Dental School, Estacio de Sa University, Rio de Janeiro, Brazil Dental School, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Medical School, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Dental School, University of Maryland Baltimore, Baltimore, Maryland, USA
Keywords chronic periodontitis, gingival crevicular fluid, HIV infections, lactoferrin, saliva. Correspondence ^nia Maria Soares Ferreira, Dr. So Educational Foundation Jayme de Altavila, ^nego Machado 918 Farol, Maceio, AL, Rua Co Brazil. Tel: +558233770028 Email: [email protected] Received 11 April 2012; accepted 30 September 2012. doi: 10.1111/jicd.12017
Abstract Aim: This study compared lactoferrin (LF) levels in the gingival crevicular fluid (GCF) and saliva between HIV-infected and noninfected patients with chronic periodontitis. Methods: For each subject, LF levels were analyzed in one shallow site (SS; PD 3 mm), one deep site (DS; PD >5 mm) and in resting whole saliva. Two groups, 28 HIV-infected and 10 noninfected, were selected. Results: Although the salivary LF levels were higher in HIV-infected than in noninfected individuals, especially in AIDS patients, this was not statistically significant (P > 0.05). Subgingival LF levels for SS and DS were lower among HIV-infected individuals, although AIDS patients showed the lowest levels. Age, smoking, gender, T CD4 lymphocytes levels and viral load did not influence subgingival LF levels, neither for SS nor for DP. Positive fungal culture was observed in 24 HIV-infected patients, but only observed in one in the control group. Overall, LF concentration was significantly higher in DS than SS, both in HIV-infected and noninfected individuals (P < 0.05) and salivary LF levels were always higher than GCF levels. Conclusion: The data indicate that LF levels in the GCF and saliva are not different between HIV-infected and noninfected patients with chronic periodontitis.
Introduction Human immunodeficiency virus (HIV) infection has been suggested as a risk factor for periodontal disease.1 Several attempts have been made in order to explain the pathogenesis of HIV-related periodontal disease, and biomarkers have been used for this purpose.2,3 It has been reported that the lactoferrin (LF) concentration in gingival crevicular fluid (GCF) shows a positive correlation with clinical parameters and it is a more sensitive indica-
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tor of periodontal pathology than traditional clinical indexes.4 Only a few controlled studies have investigated LF concentrations in HIV-infected population.5,6 Lactoferin is an iron-binding glycoprotein found in external secretions, such as saliva, milk, and tears.7,8 This glycoprotein is stored in neutrophil secondary granules7–10 and it may modulate the inflammatory response and the inhibition of cancer cell proliferation.11,12 Its main biological function is the sequestration of iron, which is required for bacterial growth.13,14 This function protects
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against oxidative stress by scavenging free iron, mainly in chronic disorders, thus altering the magnitude and the production of specific cytokines.15,16 It is also considered an important nonspecific host defense component for protection against various pathogens.16 A potent in vitro LF antiviral activity against HIV has been reported.17 In fact, LF has been studied for its antimicrobial activity against periodontopathic bacteria and its relationship with periodontal diseases.18,19 Moreover, LF also may be detected as a salivary marker and salivary LF levels have been reported as significantly lower in smokers than in noncurrent smokers with periodontitis.20 Saliva and GCF are composed of substances provenient from many sources, such as dental biofilm, inflammatory cells, host tissue, salivary glands, and serum.21–25 The collection of GCF represents a noninvasive procedure to analyse biochemical parameters of specific sites.26 Therefore, it is possible to use the GCF and saliva as sources of biomarkers associated with active periodontal disease.25,27 Since Friedman et al.28 described the importance of LF in GCF, it has been investigated as a biomarker of periodontal disease activity. However, the role of LF in GCF and saliva of HIV-infected patients is not clear.3 Therefore, the purpose of this study was to compare LF levels in GCF and saliva of HIV-infected patients with chronic periodontitis with LF levels of noninfected patients with chronic periodontitis.
Material and methods Subject population HIV-infected patients were selected from the Clinic at the University of Maryland Dental School and from the Evelyn Jordan Clinic – University of Maryland Medical School. The non-HIV-infected group was recruited from the Advanced General Clinic, University of Maryland Dental School. The study protocol was approved by the Institutional Review Board. All subjects were informed about the aim, risk, and benefits of the study, and signed a consent form. Information regarding general health and HIV-infection history was obtained from a questionnaire and the patients’ medical records. Patients to be included in the study should be over 17 years of age; have at least 20 teeth; have at least one site with probing depth (PD) 5 mm; have not used antiinflammatories and antibiotics during past 6 months; have never used antiretroviral; and have not been submited to periodontal therapy within the previous 6 months. Subjects were considered HIV-infected if seropositive in the enzyme linked immunosorbent assay (ELISA) and Western blotting tests. Noninfected individuals were considered if negative in the Western blotting saliva test. ª 2014 Wiley Publishing Asia Pty Ltd
Lactoferrin levels in HIV-infected patients
Subjects were excluded if pregnant, nursing, or presenting diabetes, autoimune diseases, aggressive periodontitis, and necrotizing periodontal disease. Subjects were distributed into two groups: HIV-infected and non-HIVinfected patients. Classification of HIV-infection was applied using the US Centers for Disease Control and Prevention (CDC) criteria.29 Briefly, patients beloging to category 1 present a T CD4 lymphocyte count 500 cells/mm3, category present a 2 T CD4 lymphocyte count between 200 and 499 cells/mm3, and category 3 present a T CD4 lymphocyte count 5 mm). For each patient, GCF was collected from one SS and one DS in order to evaluate LF levels. When there was more than one SS and DS, the sample was collected from the easier site, which presented less risk of saliva contamination. The GCF samples were collected using paper strips known as periopaper® (Harco Electronicas Irvine, CA, USA).30 Supragingival biofilm was removed and the sites were isolated with cotton rolls, in order to avoid contamination by saliva during sample collection. The strips were gently inserted and maintened into the gingival sulcus for 30 sec. Strips were then placed in sterile individual tubes with 1 mL of phosphate buffer saline (PBS) solution (pH 7.4), and was shaken at 37°C.
Saliva sampling Resting whole saliva was expectorated for 5 min and collected into a frozen tube of 50 mL. Samples were immediately placed in ice. At the laboratory samples were centrifuged at 1000 9 g for 30 min and stored at 70°C for subsequent analysis after 4–6 weeks.
Quantification of lactoferrin in saliva and GCF Lactoferrin presence and its levels were determined by a sandwich antibody assay.31 Costar Plates® (Corning Inc., Corning, NY, USA) with standardized microwells of polystyrene were utilized for this experiment. 17
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Adjustment procedure of the optical density curve X LF concentration and its estimated value as a result of the optical density The procedure was performed by the correlation between optical density and the logarithm (log) of LF concentration. This procedure consisted of adjusting calibration in pairs (optical density 9 log of LF concentration) and it utilized the curves that reached higher coefficient correlation (CC) levels – above 0.9 (the polynomial third-order curve showed CC > 0.9 and was used for all calculations). After adjustment with the polynomial curve the equation obtained allowed calculation of the log of concentration as a result of optical density. Then, the anti-log was calculated and the values of the LF concentration were determined. In order to minimize the error measurement, the mean of three measures for the same sample was calculated, so as to determine the calibrated curve and to estimate of the LF concentration. Microbiological assessment Cytological smears were obtained from the dorsum of the tongue and the buccal mucosa by exfoliative cytology. These samples were collected from normal mucosal and from oral lesions suggestive of candidiasis. Subgingival biofilm samples were colleceted using periodontal curettes from sites with PD >5 mm. When there was more than one site with PD >5 mm, the mesial sites of maxillary posterior teeth were chosen. Supragingival biofilm was removed before subgingival sampling using periodontal curettes and a rubber cup. Samples were streaked onto Sabouraud dextrose agar (SDA) plates (Difco Laboratories, Detroit, MI, USA) and incubated for 48–72 h at 35–37°C. Fungal identification Fungal species were identified using a standard mycology protocol.32,33 After 3 h of incubation at 37°C, all colony forming units (CFU) that grew in pure culture were tested for germ tube formation. The positive isolates for germ tube formation were streaked onto SDA and incubated for 48 h at 45°C. This procedure was followed in order to differentiate Candida albicans from Candida dubliniensis (C. dubliniensis does not grow at 45°C). The germ tube positive colonies that did not grow were considered suggestive of C. dubliniensis and the identification was confirmed using the coaggregation method. This is based on the capacity of C. dubliniensis to coaggregate with Fusobacterium nucleatum in vitro.34 All fungi were assessed regarding substrate assimilation using the API 20C Aux system (Biomerieux Vitek Inc., Hazelwood, MO, USA), which identifies several fungal species, including C. dubliniensis. 18
Statistical analysis All statistical tests were accomplished using the SPSS© 16.0 statistical program (SPSS Inc., IBM, Chicago, IL, USA). Significant differences in the lactoferrin concentration between and among groups were tested using the Mann– Whitney and Kruskal–Wallis tests, respectively. Significant differences within each group were determined using the Wilcoxon signed-rank test. A nonparametric test was utilized because the Kolmogorov–Smirnov test applied on lactoferrin concentration showed data that were not normally distributed. Overall, descriptive tendencies were utilized to evaluate minor differences among the groups, because sample sizes were limited. Statistical significance was reached at a 5% level for all analysis. Results Twenty-eight HIV-infected and 10 non-HIV-infected patients were selected. Clinical, demographics and fungal status of the patients are listed on Table 1. Lactoferrin concentration in total saliva The LF levels in total saliva were higher in HIV-infected subjects than in the noninfected group, although not statistically significant (P > 0.05; Table 2). Within HIVinfected individuals, the highest LF concentration was observed in AIDS patients (Table 2). When LF levels were compared among different age groups, individuals above 50 years of age presented statistically significant higher LF concentration than subjects 50 years (P < 0.05) (data not shown). In addition, although smokers and females showed LF levels higher than nonsmokers and males, respectivelly, this was not statistically significant (P > 0.05; Tables 3 and 4). Similarly, no signficant differences were observed when LF concentration in total saliva was compared between the HIV-infected and noninfected patients, either among smokers (P = 0.36) or nonsmokers (P = 0.56; Table 4). The LF concentration in saliva did not show significant differences regarding T CD4 lymphocytes levels (reference point of 200 cell/mm3) and viral load ( 0.05; Table 2). No significant differences were observed when the LF concentration in GCF was compared between subgingival shallow sites and also between deep sites regarding age (data not shown), gender, and smoking habits (P > 0.05; Tables 3 and 4). When LF levels were compared between SS and DS in HIV-positive and HIV-negative patients for the variable smoking, statistically significant differences were observed for all comparision (P < 0.05) except for non-HIV-infected smoker patients (P = 0.10; Table 4). In addition, regarding HIV-infected groups, although nonintravenous drug users demonstrated LF levels higher than intravenous drug users, this was not statistically significant (P > 0.05), and DS had statistically significant higher LF levels than SS, in both groups (P < 0.05) (data not shown). The LF concentrations in the two periodontal sites (shallow and deep) were compared among different categories of T CD4 lymphocytes levels and viral load. Patients within category 3 (AIDS) (T CD4 lymphocytes 0.05
48.3 8.5
54.3 9.8
P > 0.05
2.5 0.7
2.7 0.9
P > 0.05
2.9 0.8
3.2 1.2
P > 0.05
16 (57.1)
NA
12 (43)
1 (10)
429.6 323.2 10–1115
NA
P < 0.05
NA
NA, not applicable. †Cells per cubic millimeter of blood. ‡Values provided in copies per milliliter of blood. Not obtained in three subjects.
Positive oral fungal cultures from the dorsum of the tongue and the buccal mucosa samples were observed in 24 (85.7%) of the 28 HIV-infected patients studied (Table 1). The most frequently isolated species were C. albicans (57.1%) and C. dubliniensis (14.3%) (data not shown). There were 17 positive subgingival samples from
Table 2. Lactoferrin concentration (lg/mL) in the different collection sites among 38 patients categorized by the Centers for Disease Control and Prevention (CDC) Classification (1993) HIV ( ) (N = 10)
CDC 1/2 (N = 19)
CDC 3 (AIDS) (N = 9)
All HIV (+) (N = 28)
Collection sites
Median
Range
Median
Range
Median
Range
Median
Range
Total saliva Shallow periodontal Deep periodontal
5.2 0.06 0.4
3.9–14.5 0.01–0.2 0.1–2.4
6.2 0.06 0.6
0.6–16.5 0.01–1.0 0.02–1.2
7.2 0.02 0.3
2.0–14.8 0.01–0.2 0.02–1.9
6.7 0.05 0.4
0.6–16.5 0.01–1.0 0.02–1.9
Kruskal–Wallis test – no significant difference between groups. HIV ( ), HIV seronegative; HIV (+), HIV seropositive.
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Table 3. Lactoferrin concentration (lg/mL) in the collection sites of 38 patients according to gender Female
Males
Collection sites
Median
Range
Median
Range
Pvalue
Total saliva Shallow periodontal Deep periodontal
8.0 0.2 0.3
3.0–11.3 0.01–0.4 0.07–1.9
6.2 0.05 0.5
0.6–16.5 0.01–1.0 0.02–1.0
0.69 0.43 0.87
Mann–Whitney test – no significant difference in LF levels for each collection site between males and female.
HIV-infected patients (60.7%; Table 1), and C. albicans (47%) and C. dubliniensis (41%) were the most prevalent species (data not shown). Regarding non-HIV-infected patients only one patient presented positive oral fungal culture (C. albicans), whereas no one had a positive subgingival culture (Table 1). Discussion In the current study, no significant differences were observed in LF levels between the HIV-infected and noninfected groups, either in saliva or in subgingival sites. Despite that, salivary LF concentrations were higher in HIV-infected than in noninfected patients, and in subgin-
gival sites, there were lower GCF LF levels in HIVinfected with AIDS than in noninfected patients. These contrasting results suggest no influence of saliva LF concentration on subgingival levels. Lactoferrin levels were significantly higher in subgingival DS than in SS in patients from both groups. Previous studies have shown that LF levels are increased in inflammatory tissues.35 Neutrophils release LF in response to periodontitis inflammatory conditions. This mechanism is a potential host-defense factor against subgingival pathogenic microorganisms and may be a good marker of periodontal diseases.36 Some authors have observed that high LF levels are accompanied by an increase of neutrophil numbers and vice versa.35,37 Therefore, LF can be a marker of migration of polymorphonuclear leukocytes in the GCF.9,25,26,38–40 In addition, studies have shown that levels of GCF LF were increased in gingivitis41 and periodontitis42 sites, and after periodontal therapy its concentration significantly decreased.43 In the current study, LF levels were not evaluated after periodontal treatment, but the results demonstrated the highest GCF LF concentration in periodontitis sites. Regarding HIV-infection, the population in the present study presented a trend for lower GCF LF concentration in AIDS patients. If GCF is mainly formed by plasmatic elements and the LF concentration in GCF represents the
Table 4. Lactoferrin concentration (lg/ml) in the collected sites of the 38 studied patients, according to habit of smoking Smoker
Nonsmoker
HIV (+)
HIV ( )
HIV (+)
HIV ( )
Collection sites
Median
Range
Median
Range
P§
Median
Range
Median
Range
P-value§
Total saliva Shallow periodontal† Deep periodontal‡ P†,‡ value
7.4 0.1† 0.5‡ 0.001
0.6–16.5 0.01–1.0 0.02–1.9
4.7 0.08† 1.3‡ 0.10
3.9–8.7 0.04–0.1 0.1–1.7
0.36 0.78 0.31
5.0 0.03† 0.1‡ 0.03
2.0–13.9 0.01–0.4 0.02–1.2
5.7 0.05† 0.3‡ 0.018
3.9–14.5 0.01–0.2 0.09–2.4
0.56 0.44 0.25
†,‡Wilcoxon signed rank test – comparison between shallow and deep periodontal sites within each group. §Mann–Whitney test – comparison between HIV (+) and HIV ( ) patients.
Table 5. Lactoferrin concentration (lg/mL) in shallow and deep periodontal sites in the 38 studied patients categorized by HIV status and viral load HIV seropositive HIV seronegative
VL < 10 000 cp/mL
VL > 10 000 cp/mL
Periodontal sites
Median
Range
Median
Range
Median
Range
P-value§
Shallow† Deep‡ P †,‡ value
0.06 0.4 0.05
0.01–2.0 0.09–2.4
0.1 0.6 0.02
0.01–0.5 0.07–1.2
0.05 0.3 0.02
0.01–1.0 0.02–1.9
0.84 0.37
†,‡Wilcoxon signed rank test – comparison between shallow and deep periodontal sites. §Mann–Whitney test – comparison between viral load VL < 10 000 cp/mL and VL 10 000 cp/mL.
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levels of this protein in the plasma,41 especially in shallow periodontal sites, then the results are in agreement with those studies that described lower serological LF levels in AIDS patients.37 The small sample of AIDS individuals in the present study may explain the nonsignificant results. If this affirmative is true, then it may be suggested that there will be a reduced immune response at the subgingival site, in this population. Lactoferrin has the ability to suppress IL-1b, IL-6, and TNF-a production in mononuclear cells in response to lipopolysaccharide activation, blocking the development of inflammation.44 Moreover, it has been shown in animal studies that LF delivered at the site of injury binds to bacterial iron and lipopolysaccharide, blocking the activation of neutrophils.45 Baqui et al.30 observed that IL-1b, IL-6, and TNF-a levels in GCF of SS and DS were significantly higher in HIV-infected patients than HIV-seronegative subjects. These cytokines were found in higher levels of DS than SS. The authors concluded that high levels of these inflammatory mediators may be an important factor for the development of severe periodontitis in HIVinfected patients. Unfortunatly it was not in the scope of the present study to verify if these cytokines were related to the levels of GCF LF observed in AIDS patients of the present study. Smoking is considered a risk factor for periodontal disease.20 It has been described previously that smokers present lower LF subgingival levels than nonsmokers.46 Conversely, although data from the present study demonstrated higher LF subgingival levels in smokers than in nonsmokers, this was not statistically significant. When we compared LF levels between shallow and deep sites regarding the variable smoking, significant higher LF levels were observed in DS for all comparisons, except for HIV-seronegative smokers. In this case, it is possible that the small number of non-HIV-infected smokers (only three subjects) would be responsible for the nonsignificant results. Lactoferrin is produced predominantly by two groups of cells: myeloid and secretory epithelial cells. Regarding salivary LF levels, there were higher LF levels in the saliva of HIV-infected individuals than in the controls of the population studied, although differences were not statistically significant. Similar results were reported in others studies.5,37,47 This may suggest a subclinical glandular inflammatory disease in this popupation. High LF levels have been observed in the parotid saliva of patients with ogren’s syndrome.48,50,51 inflammatory disease48,49 and Sj€ The origin of high LF levels in these two conditions is controversial and may be due to inflammation or damage of the glandular tissue,48,50,51 suggesting that LF is delivered by degranulation of polymorphonuclear leukocytes and inflammatory stimulation of cells responsible for LF ª 2014 Wiley Publishing Asia Pty Ltd
Lactoferrin levels in HIV-infected patients
production in salivary glands. The mechanism by which salivary gland inflammation occurs in HIV-infected patients is not clear. Bacterial or viral infection and immunossupression have been suggested in the etiopathogenesis.52 The features of glandular disesase in the HIVinfected population are similar to Sj€ ogren’s syndrome, although less pronounced, indicating a similar pathogenic mechanisms, modified by the presence of HIV.52 Regarding salivary markers related to smoking, the levels of salivary LF have been demonstrated significantly lower in current smokers than in noncurrent smokers.20 Additionally, salivary LF levels were significantly decreased in active smokers in comparison to passive smokers.53 The findings for the non-HIV-infected individuals of the present study are in agreement with these results. However, when the smoker and nonsmoker HIV-infected population were compared, higher salivary LF levels were presented among the smokers. Future studies with a HIVinfected population are necessary to confirm these data and to better understand these findings. Oropharyngeal candidiasis has been correlated to high local production of LF in saliva, probably linked to leukocytes which are located in infectious sites.6 Results of the present study may also suggest this correlation because the majority (85.7%) of the 28 HIV-infected individuals in the current study presented positive oral fungal culture, mainly C. albicans, whereas only one non-HIV-infected patient presented positive oral culture. The results regarding positive oral and subgingival fungal cultures observed in the current study are in accordance with other studies.32,33 Candida albicans and C. dubliniensis also were the species more frequently found in those studies. Moreover, the increase of the prevalence of C. dubliniensis among HIV-infected individuals and its absence in non-HIV-infected individulas has been demonstrated previously,32,33 and is confirmed in the present study. Regarding age influence, there were no statiscally significant differences between shallow and deep subgingival sites when the total patients were analyzed. Similar results were encountered by Fransson et al.40 when they studied experimental gingivitis in young and elderly individuals. Nevertheless, within the HIV-seropositive group studied, patients over 50 years of age demonstrated significantly higher LF concentration than those below 50 years. This may be explained by the process of aging, which may be exacerbated in HIV-infected patients, leading to a greater delivery of LF by glandular ephitelial cells. Future investigations are necessary to better understand this aspect and the local effect of the high saliva LF levels in this population. Several studies have demonstrated a higher prevalence and severity of periodontal disease with HIV-infection,54–59 mainly in severe immunodeficiency condition (T CD4 21
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Lactoferrin levels in HIV-infected patients
lymphocytes < 200 cells/mm3).60,61 Nevertheless, others studies have reported that there is no difference in the periodontal status between HIV-seronegative and HIVseropositive individuals.62,63 In spite of the fact that immunodeficiency is an important factor related to periodontal disease in this population, some authors did not observe association between chronic periodontitis and reduced levels of T CD4 lymphocytes.64–66 Several confounding factors, such as antibiotic, antiinflammatory, and antiretroviral therapies should be controlled. In addition, immunological, metabolic, and microbiological changes in HIV-infected subjects may influence the periodontal status. However, it is not possible to control all these factors. In order to limit the possible bias related to the use of some drugs, only patients that had not utilized antiinflammatory and antibiotic drugs within the previous 6 months and never used antiretroviral drugs were included in the present study. For ethical reasons, a sample with these features is not easy to obtain. These obser-
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vations justify the small sample of individuals used in this study, especially AIDS patient. Conclusion The current study indicated that LF levels in the GCF and saliva are not different between HIV-infected and noninfected patients with chronic periodontitis. However, although the LF levels in total saliva were higher in HIVinfected patients (especially those with AIDS) than in the noninfected group, this was not statiscally significant. Conversely, the subgingival sites presented a tendency for reduction of LF concentration in patients with AIDS. Nonetheless, further investigations are required in order to obtain a final conclusion about the role of the LF levels on the pathogenesis of periodontal disease in HIVinfected population. In addition, the control group in particular was small and more studies need to be done with larger cohorts.
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21 Smith QT, Au GS, Freese PL, Osborn JB, Stoltenberg JL. Five parameters of gingival crevicular fluid from eight surfaces in periodontal health and disease. J Periodontal Res 1992; 27: 466–75. 22 Adonogianaki E, Moughat NA, Kinane DF. Lactoferrin in the gingival crevice as a marker of polymorphonuclear leucocytes in periodontal diseases. J Clin Periodontol 1993; 20: 26– 31. 23 Christodoulides N, Floriano PN, Miller CS et al. Lab-on-a-chip methods for point-of-care measurements of salivary biomarkers of periodontitis. Ann N Y Acad Sci 2007; 1098: 411 –28. 24 Zhang L, Henson BS, Camargo PM, Wong DT. The clinical value of salivary biomarkers for periodontal disease. Periodontol 2000 2009; 51: 25 –37. 25 Embery G, Waddington R. Gingival crevicular fluid: biomarkers of periodontal tissue activity. Adv Dent Res 1994; 8: 329–36. 26 Curtis MA, Gillett IR, Griffiths GS et al. Detection of high-risk groups and individuals for periodontal diseases: laboratory markers from analysis of gingival crevicular fluid. J Clin Periodontol 1989; 16: 1–11. 27 Eley BM, Cox S. Advances in periodontal diagnosis 5. Potential inflammatory and immune markers. Br Dent J 1998; 184: 220–3. 28 Friedman SA, Mandel ID, Herrera M. S. Lysozyme and lactoferrin quantitation in the crevicular fluid. J Periodontol 1983; 54: 347–50. 29 Castro KG, Ward JW, Slutsker L, Buehler JW, Jaffe HW, Berkelman RL. Revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. Morb Mortal Wkly Rep 1992; 41: RR–17. 30 Baqui AA, Meiller TF, Jabra-Rizk MA, Zhang M, Kelley JI, Falkler WA JR. Enhanced interleukin 1b, interleukin 6 and tumor necrosis factor a in gingival crevicular fluid from periodontal pockets of patients infected with human immunodeficiency virus
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1. Oral Microbiol Immunol 2000; 15: 67–73. Harlow E, Lane D. Antibodies: a laboratory manual, Cold Spring Harbor: Cold Spring Harbor Laboratory, 1988: 164. Jabra-Rizk MA, Baqui AA, Kelley JI, Falkler WA Jr, Merz WG, Meiller TF. Identification of Candida dubliniensis in a prospective study of patients in the United States. J Clin Microbiol 1999a; 37: 321–6. Jabra-Rizk MA, Ferreira SM.S, Sabet M, Falkler WA Jr, Merz WG, Meiller TF. Recovery of Candida dubliniensis and other yeast from human immunodeficiency virus-associated periodontal lesions. J Clin Microbiol 2001; 39: 4520–2. Jabra-Rizk MA, Falkler WA Jr, Merz WG, Kelley JI, Baqui AA, Meiller TF. Coaggregation of Candida dubliniensis with Fusobacterium nucleatum. J Clin Microbiol 1999b; 37: 1464–8. Nakao K, Imoto I, Ikemura N et al. Relation of lactoferrin levels in gastric mucosa with Helicobacter pylori infection and with the degree of gastric inflammation. Am J Gastroenterol 1997; 92: 1005–11. Wakabayashi H, Kondo I, Kobayashi T et al. Periodontitis, periodontopathic bacteria and lactoferrin. Biometals 2010; 23: 419–24. Van Der Strate BW, Harmsem MC, The TH et al. Plasma lactoferrin levels are decreased in end-stage AIDS patients. Viral Immunol 1999; 12: 197 –203. Fine DH, Mandel ID. Indicators of periodontal disease activity: an evaluation. J Clin Periodontol 1986; 13: 533 –46. Adonogianaki E, Moughat NA, Mooney J, Stirrups D, Kinane DF. Acutephase proteins in gingival crevicular fluid during experimentally induced gingivitis. J Periodontal Res 1994; 29: 196–202. Fransson C, Mooney J, Kinane DF, Berglundh T. Differences in the inflammatory response in young and old human subjects during the course of experimental gingivitis. J Clin Periodontol 1999; 26: 453–60.
41 Ozdemir B, Ozcan G, Karaduman B et al. Lactoferrin in gingival crevicular fluid and peripheral blood during experimental gingivitis. Eur J Dent 2009; 3: 16–23. 42 Wei P-F, Ho K-Y, Ho Y-P, Wu Y-M, Yang Y-H, Tsai CC. The investigation of glutathione peroxidase, lactoferrin, myeloperoxidase and interleukin-1b in gingival crevicular fluid: implications for oxidative stress in human periodontal diseases. J Periodont Res 2004; 39: 287–93. 43 Jentsch H, Sievert Y, G€ ocke R. Lactoferrin and other markers from gingival crevicular fluid and saliva before and after periodontal treatment. J Clin Periodontol 2004; 31: 511–4. 44 Kruzel ML, Harari Y, Mailman D, Actor JK, Zimecki M. Differential effects of prophylactic, concurrent and therapeutic lactoferrin treatment on LPS-induced inflammatory responses in mice. Clin Exp Immunol 2002; 130: 25–31. 45 Kruzel ML, Yael H, Chen C-Y, Castro GA. The gut: a key metabolic organ protected by lactoferrin during experimental systemic inflammation in mice. In: Spik G, Legrand D, Mazurier J, Pierce A, Perraudin J-P, ed. Advances in lactoferrin research, New York: Plenum Press, 1998: 167–73. 46 Persson L, Bergstrom J, Ito H, Gustafsson A. Tobacco smoking and neutrophil activity in patients with periodontal disease. J Periodontol 2001; 72: 90–5. 47 Atkinson JC, Yeh C-K, Bermudez D, Fox PC, Baum BJ. Longitudinal evaluation of major salivary gland function in HIV-1 infected patients. J Oral Pathol Med 1989; 18: 469–70. 48 Tabak L, Mandel ID, Karlan D, Baurmash H. Alterations in lactoferrin in salivary gland disease. J Dent Res 1978; 57: 43–7. 49 Benini L, Harvey RF, Vantini I et al. Lactoferrin concentration in the parotid saliva of patients with chronic pancreatitis. Digestion 1983; 28: 77– 81. 50 Zipp MM, Yasbin L, Al-Hashimi I. The effect of parotid salivary flow rate on the levels of salivary antimicrobial
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51
52
53
54
55
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proteins in patients with Sj€ ogren’s syndrome. Quintessence Int 1999; 30: 700–5. Carpenter GH, Proctor GB, Pankhurst CL, O’Donohue J, Scott D, Hunnable MP. Sialochemical markers of salivary gland involvement with Sj€ ogren’s syndrome secondary to rheumatoid arthritis and primary billary cirrhosis. J Oral Pathol Med 2000; 29: 452–9. Schiodt M, Atkinson JC, Greenspan D et al. Sialochemistry in human immunodeficiency virus associated salivary gland disease. J Rheumatol 1992; 19: 26–9. Nishida N, Yamamoto Y, Tanaka M et al. Association between passive smoking and salivary markers related to periodontitis. J Clin Periodontol 2006; 33: 717–23. Dennison DK, Smith B, Newland JR. Immune responsiveness and GUNA (abstr.). J Dent Res 1985; 64: 197. Winkler JR. AIDS virus associated periodontal disease. J Dent Res 1986; 65: 741. Massouredis CM, Katz MH, Greenspan D, Herrera C, Hollander H,
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Greenpan JS. Prevalence of HIV associated periodontitis and gingivitis in HIV infected patients attending in AIDS clinic. J Acquir Immune Defic Syndr 1992; 5: 479–83. Mckaig RG, Thomas JC, Patton LL, Strauss RP, Slade GD, Neck J.D. Prevalence of HIV associated periodontitis and chronic periodontitis in a southeastern US study group. J Public Health Dent 1998; 4: 294–300. Robinson PG, Sheiham A, Challacombe SJ, Zakrzewaska J.M. The periodontal health of homosexual men with HIV infection: a controlled study. Oral Dis 1996; 2: 45–52. Robinson PG, Adegboye A, Rowland RW, Yeung S, Johnson NW. Periodontal disease in HIV infection. Oral Dis 2002; 8: 144–50. Barr C, Lopez MR, Rua-Dobles A. Periodontal changes by HIV serostatus in a cohort of homosecual and bisexual men. J Clin Periodontol 1992; 19: 794–801. Steidley KE, Thompson SH, McQuade MJ, Strong SL, Scheidt MJ, Van Dyke A. Comparison of T4:T8 lymphocyte ratio in the periodontal
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lesion of healthy and HIV-positive patients. Lab Invest 1992; 63: 753–6. Friedman RB, Gunsolley J, Gentry A, Dinius A, Kaplowitz L, Settle J. Periodontal status of HIv seropositive and AIDS patients. J Periodontol 1991; 62: 623–7. Holmstrup P, Westergaard D. J. Periodontal diseases in HIV-infected patients. J Clin Periodontol 1994; 21: 279–80. Lucht E, Heimdahl A, Nord CE. Periodontal disease in HIV-infected patients in relation to lymphocyte subsets and specific micro-organisms. J Clin Periodontol 1991; 18: 252–6. Yeung SCH, Stewart GJ, Cooper DA, Sindhusake D. Progression of periodontal disease in HIV seropositive patients. J Periodontol 1993; 64: 651– 7. Goncßalves LdeS, Ferreira SMS, Silva A. Jr, Villoria GE, Costinha LH, Colombo AP. Association of T CD4 lymphocyte levels and chronic periodontitis in HIV-infected Brazilian patients udergoing highly active antiretroviral therapy. Clinical results. J Periodontol 2005; 76: 906–13.
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ORIGINAL ARTICLE Oral Biosciences
Lactoferrin levels in gingival crevicular fluid and saliva of HIV-infected patients with chronic periodontitis Sonia Maria Soares Ferreira1, Lucio Souza Goncßalves2,3, Sandra R. Torres4, Susie A. Nogueira5 & Timothy F. Meiller6 1 2 3 4 5 6
, Brazil Educational Foundation Jayme de Altavila, Maceio Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Dental School, Estacio de Sa University, Rio de Janeiro, Brazil Dental School, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Medical School, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Dental School, University of Maryland Baltimore, Baltimore, Maryland, USA
Keywords chronic periodontitis, gingival crevicular fluid, HIV infections, lactoferrin, saliva. Correspondence ^nia Maria Soares Ferreira, Dr. So Educational Foundation Jayme de Altavila, ^nego Machado 918 Farol, Maceio, AL, Rua Co Brazil. Tel: +558233770028 Email: [email protected] Received 11 April 2012; accepted 30 September 2012. doi: 10.1111/jicd.12017
Abstract Aim: This study compared lactoferrin (LF) levels in the gingival crevicular fluid (GCF) and saliva between HIV-infected and noninfected patients with chronic periodontitis. Methods: For each subject, LF levels were analyzed in one shallow site (SS; PD 3 mm), one deep site (DS; PD >5 mm) and in resting whole saliva. Two groups, 28 HIV-infected and 10 noninfected, were selected. Results: Although the salivary LF levels were higher in HIV-infected than in noninfected individuals, especially in AIDS patients, this was not statistically significant (P > 0.05). Subgingival LF levels for SS and DS were lower among HIV-infected individuals, although AIDS patients showed the lowest levels. Age, smoking, gender, T CD4 lymphocytes levels and viral load did not influence subgingival LF levels, neither for SS nor for DP. Positive fungal culture was observed in 24 HIV-infected patients, but only observed in one in the control group. Overall, LF concentration was significantly higher in DS than SS, both in HIV-infected and noninfected individuals (P < 0.05) and salivary LF levels were always higher than GCF levels. Conclusion: The data indicate that LF levels in the GCF and saliva are not different between HIV-infected and noninfected patients with chronic periodontitis.
Introduction Human immunodeficiency virus (HIV) infection has been suggested as a risk factor for periodontal disease.1 Several attempts have been made in order to explain the pathogenesis of HIV-related periodontal disease, and biomarkers have been used for this purpose.2,3 It has been reported that the lactoferrin (LF) concentration in gingival crevicular fluid (GCF) shows a positive correlation with clinical parameters and it is a more sensitive indica-
16
tor of periodontal pathology than traditional clinical indexes.4 Only a few controlled studies have investigated LF concentrations in HIV-infected population.5,6 Lactoferin is an iron-binding glycoprotein found in external secretions, such as saliva, milk, and tears.7,8 This glycoprotein is stored in neutrophil secondary granules7–10 and it may modulate the inflammatory response and the inhibition of cancer cell proliferation.11,12 Its main biological function is the sequestration of iron, which is required for bacterial growth.13,14 This function protects
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against oxidative stress by scavenging free iron, mainly in chronic disorders, thus altering the magnitude and the production of specific cytokines.15,16 It is also considered an important nonspecific host defense component for protection against various pathogens.16 A potent in vitro LF antiviral activity against HIV has been reported.17 In fact, LF has been studied for its antimicrobial activity against periodontopathic bacteria and its relationship with periodontal diseases.18,19 Moreover, LF also may be detected as a salivary marker and salivary LF levels have been reported as significantly lower in smokers than in noncurrent smokers with periodontitis.20 Saliva and GCF are composed of substances provenient from many sources, such as dental biofilm, inflammatory cells, host tissue, salivary glands, and serum.21–25 The collection of GCF represents a noninvasive procedure to analyse biochemical parameters of specific sites.26 Therefore, it is possible to use the GCF and saliva as sources of biomarkers associated with active periodontal disease.25,27 Since Friedman et al.28 described the importance of LF in GCF, it has been investigated as a biomarker of periodontal disease activity. However, the role of LF in GCF and saliva of HIV-infected patients is not clear.3 Therefore, the purpose of this study was to compare LF levels in GCF and saliva of HIV-infected patients with chronic periodontitis with LF levels of noninfected patients with chronic periodontitis.
Material and methods Subject population HIV-infected patients were selected from the Clinic at the University of Maryland Dental School and from the Evelyn Jordan Clinic – University of Maryland Medical School. The non-HIV-infected group was recruited from the Advanced General Clinic, University of Maryland Dental School. The study protocol was approved by the Institutional Review Board. All subjects were informed about the aim, risk, and benefits of the study, and signed a consent form. Information regarding general health and HIV-infection history was obtained from a questionnaire and the patients’ medical records. Patients to be included in the study should be over 17 years of age; have at least 20 teeth; have at least one site with probing depth (PD) 5 mm; have not used antiinflammatories and antibiotics during past 6 months; have never used antiretroviral; and have not been submited to periodontal therapy within the previous 6 months. Subjects were considered HIV-infected if seropositive in the enzyme linked immunosorbent assay (ELISA) and Western blotting tests. Noninfected individuals were considered if negative in the Western blotting saliva test. ª 2014 Wiley Publishing Asia Pty Ltd
Lactoferrin levels in HIV-infected patients
Subjects were excluded if pregnant, nursing, or presenting diabetes, autoimune diseases, aggressive periodontitis, and necrotizing periodontal disease. Subjects were distributed into two groups: HIV-infected and non-HIVinfected patients. Classification of HIV-infection was applied using the US Centers for Disease Control and Prevention (CDC) criteria.29 Briefly, patients beloging to category 1 present a T CD4 lymphocyte count 500 cells/mm3, category present a 2 T CD4 lymphocyte count between 200 and 499 cells/mm3, and category 3 present a T CD4 lymphocyte count 5 mm). For each patient, GCF was collected from one SS and one DS in order to evaluate LF levels. When there was more than one SS and DS, the sample was collected from the easier site, which presented less risk of saliva contamination. The GCF samples were collected using paper strips known as periopaper® (Harco Electronicas Irvine, CA, USA).30 Supragingival biofilm was removed and the sites were isolated with cotton rolls, in order to avoid contamination by saliva during sample collection. The strips were gently inserted and maintened into the gingival sulcus for 30 sec. Strips were then placed in sterile individual tubes with 1 mL of phosphate buffer saline (PBS) solution (pH 7.4), and was shaken at 37°C.
Saliva sampling Resting whole saliva was expectorated for 5 min and collected into a frozen tube of 50 mL. Samples were immediately placed in ice. At the laboratory samples were centrifuged at 1000 9 g for 30 min and stored at 70°C for subsequent analysis after 4–6 weeks.
Quantification of lactoferrin in saliva and GCF Lactoferrin presence and its levels were determined by a sandwich antibody assay.31 Costar Plates® (Corning Inc., Corning, NY, USA) with standardized microwells of polystyrene were utilized for this experiment. 17
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Lactoferrin levels in HIV-infected patients
Adjustment procedure of the optical density curve X LF concentration and its estimated value as a result of the optical density The procedure was performed by the correlation between optical density and the logarithm (log) of LF concentration. This procedure consisted of adjusting calibration in pairs (optical density 9 log of LF concentration) and it utilized the curves that reached higher coefficient correlation (CC) levels – above 0.9 (the polynomial third-order curve showed CC > 0.9 and was used for all calculations). After adjustment with the polynomial curve the equation obtained allowed calculation of the log of concentration as a result of optical density. Then, the anti-log was calculated and the values of the LF concentration were determined. In order to minimize the error measurement, the mean of three measures for the same sample was calculated, so as to determine the calibrated curve and to estimate of the LF concentration. Microbiological assessment Cytological smears were obtained from the dorsum of the tongue and the buccal mucosa by exfoliative cytology. These samples were collected from normal mucosal and from oral lesions suggestive of candidiasis. Subgingival biofilm samples were colleceted using periodontal curettes from sites with PD >5 mm. When there was more than one site with PD >5 mm, the mesial sites of maxillary posterior teeth were chosen. Supragingival biofilm was removed before subgingival sampling using periodontal curettes and a rubber cup. Samples were streaked onto Sabouraud dextrose agar (SDA) plates (Difco Laboratories, Detroit, MI, USA) and incubated for 48–72 h at 35–37°C. Fungal identification Fungal species were identified using a standard mycology protocol.32,33 After 3 h of incubation at 37°C, all colony forming units (CFU) that grew in pure culture were tested for germ tube formation. The positive isolates for germ tube formation were streaked onto SDA and incubated for 48 h at 45°C. This procedure was followed in order to differentiate Candida albicans from Candida dubliniensis (C. dubliniensis does not grow at 45°C). The germ tube positive colonies that did not grow were considered suggestive of C. dubliniensis and the identification was confirmed using the coaggregation method. This is based on the capacity of C. dubliniensis to coaggregate with Fusobacterium nucleatum in vitro.34 All fungi were assessed regarding substrate assimilation using the API 20C Aux system (Biomerieux Vitek Inc., Hazelwood, MO, USA), which identifies several fungal species, including C. dubliniensis. 18
Statistical analysis All statistical tests were accomplished using the SPSS© 16.0 statistical program (SPSS Inc., IBM, Chicago, IL, USA). Significant differences in the lactoferrin concentration between and among groups were tested using the Mann– Whitney and Kruskal–Wallis tests, respectively. Significant differences within each group were determined using the Wilcoxon signed-rank test. A nonparametric test was utilized because the Kolmogorov–Smirnov test applied on lactoferrin concentration showed data that were not normally distributed. Overall, descriptive tendencies were utilized to evaluate minor differences among the groups, because sample sizes were limited. Statistical significance was reached at a 5% level for all analysis. Results Twenty-eight HIV-infected and 10 non-HIV-infected patients were selected. Clinical, demographics and fungal status of the patients are listed on Table 1. Lactoferrin concentration in total saliva The LF levels in total saliva were higher in HIV-infected subjects than in the noninfected group, although not statistically significant (P > 0.05; Table 2). Within HIVinfected individuals, the highest LF concentration was observed in AIDS patients (Table 2). When LF levels were compared among different age groups, individuals above 50 years of age presented statistically significant higher LF concentration than subjects 50 years (P < 0.05) (data not shown). In addition, although smokers and females showed LF levels higher than nonsmokers and males, respectivelly, this was not statistically significant (P > 0.05; Tables 3 and 4). Similarly, no signficant differences were observed when LF concentration in total saliva was compared between the HIV-infected and noninfected patients, either among smokers (P = 0.36) or nonsmokers (P = 0.56; Table 4). The LF concentration in saliva did not show significant differences regarding T CD4 lymphocytes levels (reference point of 200 cell/mm3) and viral load ( 0.05; Table 2). No significant differences were observed when the LF concentration in GCF was compared between subgingival shallow sites and also between deep sites regarding age (data not shown), gender, and smoking habits (P > 0.05; Tables 3 and 4). When LF levels were compared between SS and DS in HIV-positive and HIV-negative patients for the variable smoking, statistically significant differences were observed for all comparision (P < 0.05) except for non-HIV-infected smoker patients (P = 0.10; Table 4). In addition, regarding HIV-infected groups, although nonintravenous drug users demonstrated LF levels higher than intravenous drug users, this was not statistically significant (P > 0.05), and DS had statistically significant higher LF levels than SS, in both groups (P < 0.05) (data not shown). The LF concentrations in the two periodontal sites (shallow and deep) were compared among different categories of T CD4 lymphocytes levels and viral load. Patients within category 3 (AIDS) (T CD4 lymphocytes 0.05
48.3 8.5
54.3 9.8
P > 0.05
2.5 0.7
2.7 0.9
P > 0.05
2.9 0.8
3.2 1.2
P > 0.05
16 (57.1)
NA
12 (43)
1 (10)
429.6 323.2 10–1115
NA
P < 0.05
NA
NA, not applicable. †Cells per cubic millimeter of blood. ‡Values provided in copies per milliliter of blood. Not obtained in three subjects.
Positive oral fungal cultures from the dorsum of the tongue and the buccal mucosa samples were observed in 24 (85.7%) of the 28 HIV-infected patients studied (Table 1). The most frequently isolated species were C. albicans (57.1%) and C. dubliniensis (14.3%) (data not shown). There were 17 positive subgingival samples from
Table 2. Lactoferrin concentration (lg/mL) in the different collection sites among 38 patients categorized by the Centers for Disease Control and Prevention (CDC) Classification (1993) HIV ( ) (N = 10)
CDC 1/2 (N = 19)
CDC 3 (AIDS) (N = 9)
All HIV (+) (N = 28)
Collection sites
Median
Range
Median
Range
Median
Range
Median
Range
Total saliva Shallow periodontal Deep periodontal
5.2 0.06 0.4
3.9–14.5 0.01–0.2 0.1–2.4
6.2 0.06 0.6
0.6–16.5 0.01–1.0 0.02–1.2
7.2 0.02 0.3
2.0–14.8 0.01–0.2 0.02–1.9
6.7 0.05 0.4
0.6–16.5 0.01–1.0 0.02–1.9
Kruskal–Wallis test – no significant difference between groups. HIV ( ), HIV seronegative; HIV (+), HIV seropositive.
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S.M. Soares Ferreira et al.
Lactoferrin levels in HIV-infected patients
Table 3. Lactoferrin concentration (lg/mL) in the collection sites of 38 patients according to gender Female
Males
Collection sites
Median
Range
Median
Range
Pvalue
Total saliva Shallow periodontal Deep periodontal
8.0 0.2 0.3
3.0–11.3 0.01–0.4 0.07–1.9
6.2 0.05 0.5
0.6–16.5 0.01–1.0 0.02–1.0
0.69 0.43 0.87
Mann–Whitney test – no significant difference in LF levels for each collection site between males and female.
HIV-infected patients (60.7%; Table 1), and C. albicans (47%) and C. dubliniensis (41%) were the most prevalent species (data not shown). Regarding non-HIV-infected patients only one patient presented positive oral fungal culture (C. albicans), whereas no one had a positive subgingival culture (Table 1). Discussion In the current study, no significant differences were observed in LF levels between the HIV-infected and noninfected groups, either in saliva or in subgingival sites. Despite that, salivary LF concentrations were higher in HIV-infected than in noninfected patients, and in subgin-
gival sites, there were lower GCF LF levels in HIVinfected with AIDS than in noninfected patients. These contrasting results suggest no influence of saliva LF concentration on subgingival levels. Lactoferrin levels were significantly higher in subgingival DS than in SS in patients from both groups. Previous studies have shown that LF levels are increased in inflammatory tissues.35 Neutrophils release LF in response to periodontitis inflammatory conditions. This mechanism is a potential host-defense factor against subgingival pathogenic microorganisms and may be a good marker of periodontal diseases.36 Some authors have observed that high LF levels are accompanied by an increase of neutrophil numbers and vice versa.35,37 Therefore, LF can be a marker of migration of polymorphonuclear leukocytes in the GCF.9,25,26,38–40 In addition, studies have shown that levels of GCF LF were increased in gingivitis41 and periodontitis42 sites, and after periodontal therapy its concentration significantly decreased.43 In the current study, LF levels were not evaluated after periodontal treatment, but the results demonstrated the highest GCF LF concentration in periodontitis sites. Regarding HIV-infection, the population in the present study presented a trend for lower GCF LF concentration in AIDS patients. If GCF is mainly formed by plasmatic elements and the LF concentration in GCF represents the
Table 4. Lactoferrin concentration (lg/ml) in the collected sites of the 38 studied patients, according to habit of smoking Smoker
Nonsmoker
HIV (+)
HIV ( )
HIV (+)
HIV ( )
Collection sites
Median
Range
Median
Range
P§
Median
Range
Median
Range
P-value§
Total saliva Shallow periodontal† Deep periodontal‡ P†,‡ value
7.4 0.1† 0.5‡ 0.001
0.6–16.5 0.01–1.0 0.02–1.9
4.7 0.08† 1.3‡ 0.10
3.9–8.7 0.04–0.1 0.1–1.7
0.36 0.78 0.31
5.0 0.03† 0.1‡ 0.03
2.0–13.9 0.01–0.4 0.02–1.2
5.7 0.05† 0.3‡ 0.018
3.9–14.5 0.01–0.2 0.09–2.4
0.56 0.44 0.25
†,‡Wilcoxon signed rank test – comparison between shallow and deep periodontal sites within each group. §Mann–Whitney test – comparison between HIV (+) and HIV ( ) patients.
Table 5. Lactoferrin concentration (lg/mL) in shallow and deep periodontal sites in the 38 studied patients categorized by HIV status and viral load HIV seropositive HIV seronegative
VL < 10 000 cp/mL
VL > 10 000 cp/mL
Periodontal sites
Median
Range
Median
Range
Median
Range
P-value§
Shallow† Deep‡ P †,‡ value
0.06 0.4 0.05
0.01–2.0 0.09–2.4
0.1 0.6 0.02
0.01–0.5 0.07–1.2
0.05 0.3 0.02
0.01–1.0 0.02–1.9
0.84 0.37
†,‡Wilcoxon signed rank test – comparison between shallow and deep periodontal sites. §Mann–Whitney test – comparison between viral load VL < 10 000 cp/mL and VL 10 000 cp/mL.
20
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S.M. Soares Ferreira et al.
levels of this protein in the plasma,41 especially in shallow periodontal sites, then the results are in agreement with those studies that described lower serological LF levels in AIDS patients.37 The small sample of AIDS individuals in the present study may explain the nonsignificant results. If this affirmative is true, then it may be suggested that there will be a reduced immune response at the subgingival site, in this population. Lactoferrin has the ability to suppress IL-1b, IL-6, and TNF-a production in mononuclear cells in response to lipopolysaccharide activation, blocking the development of inflammation.44 Moreover, it has been shown in animal studies that LF delivered at the site of injury binds to bacterial iron and lipopolysaccharide, blocking the activation of neutrophils.45 Baqui et al.30 observed that IL-1b, IL-6, and TNF-a levels in GCF of SS and DS were significantly higher in HIV-infected patients than HIV-seronegative subjects. These cytokines were found in higher levels of DS than SS. The authors concluded that high levels of these inflammatory mediators may be an important factor for the development of severe periodontitis in HIVinfected patients. Unfortunatly it was not in the scope of the present study to verify if these cytokines were related to the levels of GCF LF observed in AIDS patients of the present study. Smoking is considered a risk factor for periodontal disease.20 It has been described previously that smokers present lower LF subgingival levels than nonsmokers.46 Conversely, although data from the present study demonstrated higher LF subgingival levels in smokers than in nonsmokers, this was not statistically significant. When we compared LF levels between shallow and deep sites regarding the variable smoking, significant higher LF levels were observed in DS for all comparisons, except for HIV-seronegative smokers. In this case, it is possible that the small number of non-HIV-infected smokers (only three subjects) would be responsible for the nonsignificant results. Lactoferrin is produced predominantly by two groups of cells: myeloid and secretory epithelial cells. Regarding salivary LF levels, there were higher LF levels in the saliva of HIV-infected individuals than in the controls of the population studied, although differences were not statistically significant. Similar results were reported in others studies.5,37,47 This may suggest a subclinical glandular inflammatory disease in this popupation. High LF levels have been observed in the parotid saliva of patients with ogren’s syndrome.48,50,51 inflammatory disease48,49 and Sj€ The origin of high LF levels in these two conditions is controversial and may be due to inflammation or damage of the glandular tissue,48,50,51 suggesting that LF is delivered by degranulation of polymorphonuclear leukocytes and inflammatory stimulation of cells responsible for LF ª 2014 Wiley Publishing Asia Pty Ltd
Lactoferrin levels in HIV-infected patients
production in salivary glands. The mechanism by which salivary gland inflammation occurs in HIV-infected patients is not clear. Bacterial or viral infection and immunossupression have been suggested in the etiopathogenesis.52 The features of glandular disesase in the HIVinfected population are similar to Sj€ ogren’s syndrome, although less pronounced, indicating a similar pathogenic mechanisms, modified by the presence of HIV.52 Regarding salivary markers related to smoking, the levels of salivary LF have been demonstrated significantly lower in current smokers than in noncurrent smokers.20 Additionally, salivary LF levels were significantly decreased in active smokers in comparison to passive smokers.53 The findings for the non-HIV-infected individuals of the present study are in agreement with these results. However, when the smoker and nonsmoker HIV-infected population were compared, higher salivary LF levels were presented among the smokers. Future studies with a HIVinfected population are necessary to confirm these data and to better understand these findings. Oropharyngeal candidiasis has been correlated to high local production of LF in saliva, probably linked to leukocytes which are located in infectious sites.6 Results of the present study may also suggest this correlation because the majority (85.7%) of the 28 HIV-infected individuals in the current study presented positive oral fungal culture, mainly C. albicans, whereas only one non-HIV-infected patient presented positive oral culture. The results regarding positive oral and subgingival fungal cultures observed in the current study are in accordance with other studies.32,33 Candida albicans and C. dubliniensis also were the species more frequently found in those studies. Moreover, the increase of the prevalence of C. dubliniensis among HIV-infected individuals and its absence in non-HIV-infected individulas has been demonstrated previously,32,33 and is confirmed in the present study. Regarding age influence, there were no statiscally significant differences between shallow and deep subgingival sites when the total patients were analyzed. Similar results were encountered by Fransson et al.40 when they studied experimental gingivitis in young and elderly individuals. Nevertheless, within the HIV-seropositive group studied, patients over 50 years of age demonstrated significantly higher LF concentration than those below 50 years. This may be explained by the process of aging, which may be exacerbated in HIV-infected patients, leading to a greater delivery of LF by glandular ephitelial cells. Future investigations are necessary to better understand this aspect and the local effect of the high saliva LF levels in this population. Several studies have demonstrated a higher prevalence and severity of periodontal disease with HIV-infection,54–59 mainly in severe immunodeficiency condition (T CD4 21
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Lactoferrin levels in HIV-infected patients
lymphocytes < 200 cells/mm3).60,61 Nevertheless, others studies have reported that there is no difference in the periodontal status between HIV-seronegative and HIVseropositive individuals.62,63 In spite of the fact that immunodeficiency is an important factor related to periodontal disease in this population, some authors did not observe association between chronic periodontitis and reduced levels of T CD4 lymphocytes.64–66 Several confounding factors, such as antibiotic, antiinflammatory, and antiretroviral therapies should be controlled. In addition, immunological, metabolic, and microbiological changes in HIV-infected subjects may influence the periodontal status. However, it is not possible to control all these factors. In order to limit the possible bias related to the use of some drugs, only patients that had not utilized antiinflammatory and antibiotic drugs within the previous 6 months and never used antiretroviral drugs were included in the present study. For ethical reasons, a sample with these features is not easy to obtain. These obser-
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vations justify the small sample of individuals used in this study, especially AIDS patient. Conclusion The current study indicated that LF levels in the GCF and saliva are not different between HIV-infected and noninfected patients with chronic periodontitis. However, although the LF levels in total saliva were higher in HIVinfected patients (especially those with AIDS) than in the noninfected group, this was not statiscally significant. Conversely, the subgingival sites presented a tendency for reduction of LF concentration in patients with AIDS. Nonetheless, further investigations are required in order to obtain a final conclusion about the role of the LF levels on the pathogenesis of periodontal disease in HIVinfected population. In addition, the control group in particular was small and more studies need to be done with larger cohorts.
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