*Agnieszka Bogusławska-Kapała1, 2, Agnieszka Piekarska3, Jolanta Ochocińska1, Alicja Cackowska-Lass1, Agata Żółtowska1, Barbara Kochańska1
An evaluation of salivary levels of lysozyme and lactoferrin in patients in the late period after allogeneic hematopoietic stem cell transplantation (HSCT)
Ocena stężenia lizozymu i laktoferyny w ślinie pacjentów będących w późnym okresie po allogenicznej transplantacji komórek hematopoezy (HCT)
1Department of Conservative Dentistry, Medical University of Gdańsk
Head of Department: Associate Professor Barbara Kochańska, MD, PhD
2Department of Integrated Dentistry, Department of Conservative Dentistry, Medical University of Warsaw
Head of Department: Izabela Strużycka, MD, PhD
3Department of Hematology and Transplantation, Medical University of Gdańsk
Head of Department: Professor Andrzej Hellman, MD, PhD
Wstęp. Transplantacja komórek hematopoezy (ang. hematopoietic cell transplantation – HCT) często łączy się z występowaniem powikłań w jamie ustnej, niejednokrotnie zagrażających zdrowiu i życiu pacjentów. Zaburzenia jakościowe i ilościowe czynników nieswoistej odporności zawartych w ślinie, takich jak lizozym i laktoferyna, mogą przyczyniać się do rozwoju tych powikłań.
Cel pracy. Analiza stężenia lizozymu i laktoferyny w ślinie pacjentów będących w późnym okresie (powyżej setnej doby) po allogenicznej transplantacji komórek krwiotwórczych, z uwzględnieniem czasu, jaki upłynął od transplantacji oraz szybkości wydzielania śliny.
Materiał i metody. Zbadano 45 osób będących 3,5 miesiąca do 5 lat po HCT. Do oznaczenia stężenia lizozymu i laktoferyny w ślinie spoczynkowej i stymulowanej zastosowano metodę ELISA (ang. enzyme-linked immunosorbent assay).
Wyniki. Średnie stężenie lizozymu i laktoferyny w ślinie nie zmieniało się istotnie w zależności od upływu czasu po przeszczepieniu. Obserwowano znaczne wahania stężenia lizozymu. Nie stwierdzono zależności pomiędzy stężeniem lizozymu a szybkością wydzielania śliny. Średnie stężenie laktoferyny było istotnie statystycznie wyższe w grupie pacjentów po HCT w porównaniu z grupą kontrolną i wzrastało istotnie wraz ze spadkiem szybkości wydzielania śliny spoczynkowej i stymulowanej.
Wnioski. W każdym okresie po HCT należy spodziewać się znaczących wahań stężenia czynników odpornościowych zawartych w ślinie, szczególnie u pacjentów poddawanych immunosupresji, z zaburzeniami wydzielania śliny i/lub z chorobą „przeszczep przeciwko gospodarzowi”. Wysokie stężenie laktoferyny w ślinie może stanowić wskaźnik stanu zapalnego w obrębie tkanek miękkich jamy ustnej.
Introduction. Hematopoietic Stem Cell Transplantation (HSCT) is often associated with oral complications, which frequently affect the health and even lives of patients. Quantitative and qualitative impairment of salivary immunological factors, such as lactoferrin and lysozyme, can contribute to the development of these complications.
Aim. An analysis of salivary lysozyme and lactoferrin levels in patients in the late period (more than 100 days) after allogeneic stem cell transplantation. The time elapsed since transplantation and salivary flow rate were also taken into consideration.
Material and methods. A total of 45 patients 3.5 months to 5 years after HSCT were evaluated. Enzyme-linked immunosorbent assay (ELISA) was used to determine saliva lactoferrin and lysozyme levels.
Results. No significant relationship was found between mean saliva lysozyme and lactoferrin levels and the time elapsed since HSCT. Significant differences in lysozyme levels were observed. No correlation was found between lysozyme levels and salivary flow rate. Mean lactoferrin levels were statistically significantly higher in post-HSCT patients compared to the control group and increased with a decrease in the stimulated and non-stimulated salivary flow rate.
Conclusions. Significant variations in the levels of immunological salivary factors should be always expected after HSCT, particularly in patients under immunosuppression and/or those with graft-versus-host disease. High levels of salivary lactoferrin can be an indicator of oral inflammation.
Hematopoietic stem cell transplantation (HSCT) is an increasingly used therapeutic method in a number of diseases, such as hematopoietic proliferative diseases and congenital metabolic and immune disorders (1).
Patient’s own cells (autologous HSCT) or donor’s cells (allogeneic HSCT) are used for transplantation. The regeneration of hematopoietic system after allo-HSCT is a slow process. Quantitative and qualitative impairment of cellular and humoral immunity persists for a long time in most patients (2). Despite significant therapeutic advances, hematopoietic stem cell transplantation involves the risk of a number of complications, which also affect the oral cavity in most patients (2, 3). The most serious complications include inflammation of the oral mucosa and reduced saliva production (3, 4). Reduced salivary secretion leads to changes in saliva composition, including altered levels of nonspecific and specific immune factors (5-7), which may in turn significantly contribute to the development of pathological processes in the oral cavity (8).
Lysozyme and lactoferrin belong to the most important nonspecific salivary immune factors (8-10). Lysozyme (muramidase) is a cationic protein found in the secretion from the serous cells of the salivary glands, gingival crevicular fluid as well as neutrophils, monocytes and macrophages. The enzyme has a broad spectrum of non-specific action on pathogenic microorganisms. It acts primarily on gram-positive bacteria (Streptococcus mutans, Lactobacillus acidophilus), to a lesser degree on gram-negative bacteria (e.g. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis) and some fungal species (e.g. Candida spp.) (3, 11). Lysozyme causes microbial cell lysis by hydrolysing the bonds between N-acetylglucosamine and N-acetylmuramic acid in cellular wall peptidoglycan (12). Additionally, it can activate bacterial autolysis as well as inhibit bacterial glucose absorption and acid production (11, 12). It was found that lysozyme can also induce microbial aggregation and inactivate some viruses (11). Muramidase also acts as an anti-inflammatory agent by the inhibition of leukocyte chemotaxis and a direct modulation of complement reaction (10). Additionally, the agent is involved in the production of gamma globulins, it accelerates granulation tissue formation (12) and has anticancer properties (13).
Lactoferrin is a glycoprotein belonging to the transferrin family, produced by epithelial cells, which is very common in secretory fluids such as milk, saliva, tears or gastrointestinal secretion (14). It is also one of the main components in the granules of neutrophils, from which it is released during inflammation (15), therefore an increased level of this protein is considered by some authors to be an important indicator of inflammation (9, 15). Salivary lactoferrin is produced in the epithelial cells of the serous salivary glands. It may be also derived from plasma penetrating into saliva during inflammation of the mucosa and/or salivary glands (6) or form as a product of degradation of granulocytes located in the gingival sulcus (4). Lactoferrin has biostatic and biocidal effects against a number of bacteria (including S. mutans, S. mitis, S. salivarius), fungi (C. albicans) and viruses (HPV1) (9, 16). The antimicrobial activity of lactoferrin is related to e.g. its high capacity of binding iron ions, and thus depriving pathogens of this element (13, 17). Additionally, when degraded by pepsin, lactoferrin releases peptides showing direct bacteriostatic and antifungal activity (12). Lactoferrin also exerts immunomodulating effects, e.g. by stimulating lymphocytes to increase TNF-α and INF-γ cytokine production as well as by stimulating neutrophil phagocytosis and the release of interleukin (Il)-8 (18). Furthermore, lactoferrin regulates the production of GM-CSF (granulocyte-macrophage colony-stimulating factor) in macrophages (14) and impairs microbial adhesion to tissues, including the adhesion of Streptococcus mutans to tooth enamel hydroxyapatite (18).
The aim of the study was to analyse the levels of selected non-specific immune components of mixed saliva, i.e. lysozyme and lactoferrin, in patients in the late period (i.e. more than 100 days) after allogeneic hematopoietic cell transplantation. It was also evaluated whether there were significant differences in mixed saliva levels of lysozyme and lactoferrin, depending on the time elapsed since transplantation. Furthermore, the relationship between mixed saliva levels of lysozyme and lactoferrin and the salivary flow rate was assessed.
Material and methods
A total of 45 patients (17 women and 28 men) 3.5 months up to 5 years after allogeneic HSCT, remaining under the care of the Department of Haematology and Transplantation at the Medical University of Gdańsk and the Department of Conservative Dentistry at the Medical University of Gdańsk were included in the study. The study was approved by the Bioethics Committee of the Medical University of Gdańsk (No. NKEBN/886/2004). All tests were performed in accordance with the recommendations of the Helsinki Convention.
Patients were divided into three groups, depending on the time elapsed since transplantation, to include the gradual process of hematopoietic reconstitution after allogeneic HSTC in the assessment. Group I included patients 3.5 to 10 months after transplantation (i.e. in the phase of significant hematopoietic immaturity). Group II included patients 12 to 24 months after transplantation (i.e. in the phase of progressive stabilization of the hematopoietic system). Group III included patients more than 24 months after transplantation (i.e. in the phase of full hematopoietic maturity). Control group included 27 healthy individuals who consented to participate in the study (tab. 1).
Tab. 1. Patient characteristics, taking into account the time elapsed since allogeneic HSCT
|Group||Number of subjects||Time since allo-HSCT (months)||Age (years)|
|n||? ± δ||Me||range||? ± δ||Me||range|
|I||20||5.9 ± 2.3||6||3.5-10||41.6 ± 10.4||46||22-54|
|II||14||19.1 ± 3.2||18.5||12-24||31.4 ± 7.7||30||21-46|
|III||11||36.9 ± 10.8||36||27-66||40.4 ± 10.8||40||19-54|
|In total||45||17.6 ± 13.7||17||3.5-66||38.1 ± 10.1||38||19-54|
Stimulated and resting mixed saliva was taken from all patients before noon (the patients refrained from eating, tooth brushing, smoking, chewing gum, and also drinking, unless precluded by their general condition or the degree of mouth dryness, for two hours before taking samples). Collection of resting saliva involved 2-minute salivary accumulation in the mouth followed by spitting into a calibrated test tube of a Corning type. This activity was repeated three times, which gave the total time of saliva collection equal to 6 minutes. The total volume of the collected resting saliva was divided by 6 to obtain the amount of saliva secreted during one minute (ml/min). Salivary stimulation involved chewing a paraffin block for 6 minutes. The first portion of saliva (after 1-minute stimulation) was swallowed by the patient, while the subsequent portions, secreted during the remaining 5 minutes of chewing, were collected in the test tube. Next, the amount secreted during one minute (ml/min) was calculated based on the total volume of the collected stimulated saliva. Lactoferrin and lysozyme levels were assayed in the centrifuged mixed resting and stimulated saliva (10,000 x g; 10 min). Biochemical salivary testing was performed in the laboratory of the Department of Conservative Dentistry at the Medical University of Gdańsk. A two-step method based on ELISA was used for lysozyme and lactoferrin level assessment (19). Human milk lysozyme (Sigma-Aldrich) was used as a reference. The obtained results are given in μg/ml. The non-parametric Mann-Whitney U test and Spearman’ rank correlation were used in statistical analysis. The level of significance was p < 0.05.
Table 2 shows lysozyme levels and table 3 shows lactoferrin levels in mixed resting and stimulated saliva from 45 patients after allogeneic HSCT and 27 controls. The analysis of salivary parameters in post-transplant patients revealed that the mean lysozyme and lactoferrin levels in mixed resting and stimulated saliva did not change significantly depending on the time elapsed since transplantation.
Tab. 2. Resting and stimulated salivary levels of lysozyme in terms of the time elapsed since allogeneic HSCT as well as the salivary flow rate vs. control group
|Study group||Number of subjects||Time since allo-HSCT (months)||Lysozyme levels|
|? ± δ||Me||range||? ± δ||Me||range|
|I||20||3.5-10||19.5 ± 23.9||12.4||0.8-101.0||20.4 ± 31.6||10.7||3.1-124.8|
|II||14||12-24||11.2 ± 7.9||9.9||0.42-21.9||12.0 ± 8.7||9.2||2.4-33.2|
|III||11||27-66||10.1 ± ±5.8||8.5||1.86-16.3||16.4 ± 10.5||19.2||0.54-33.1|
|In total||45||3.5-66||14.6 ± 17.6||10.9||0.42-101.0||16.8 ± 22.2||11.0||0.54-124.8|
|Controls||27||–||12.8 ± 9.5||10.3||1.1-24.7||10.4 ± 7.8||7.7||0.49-27.48|
|Correlation between salivary levels of lysozyme and salivary flow rate|
|r = -0.078; p = 0.608 (NS)||r = -0.091; p = 0.553 (NS)|
|Controls||r = 0.063; p = 0.751 (NS)||r = -0.061; p = 0.758 (NS)|
|Correlation between salivary levels of lysozyme and time since allo-HSCT ||r = -0.120; p = 0.431 (NS)||r = 0.053; p = 0.729 (NS)|
The significance of differences was evaluated based on the Mann-Whitney U test
Spearman’s correlation coefficient, NS – insignificant correlation
Tab. 3. An assessment of resting and stimulated salivary levels of lactoferrin in terms of the time elapsed since allogeneic HSCT as well as the salivary flow rate vs. control group
|Study group||Number of subjects||Time since allo-HSCT (months)||Lactoferrin levels|
|? ± δ||Me||range||? ± δ||Me||range|
|I||20||3.5-10||46.1 ± 49.0c||23.9||0.83-169.3||29.4 ± 22.6d||26.8||0.99-96.5|
|II||14||12-24||28.5 ± 30.4 ||19.9||3.4-116.8||21.4 ± 26.5||13.7||3.9-105.9|
|III||11||27-66||38.5 ± 35.0e||27.0||5.91-96.0||30.4 ± 28.5f||25.2||2.0-65.9|
|In total||45||3.5-66||38.8 ± 40.6a||22.8||0.83-169.3||27.2 ± 25.1b||24.6||0.99-105.9|
|Controls||27||–||18.9 ± 30.3a, c, e||11.2||0.62-147.5||10.5 ± 13.8b, d, f||7.1||1.8-74.5|
|Correlation between salivary levels of lactoferrin and salivary flow rate ||Post-allo-HSCT patients||r = -0.290; p < 0.05*||r = -0.350; p < 0.05*|
|Controls||r = -0.393; p < 0.05*||r = -0.780; p = 0.371 (NS)|
|Correlation between salivary levels of lactoferrin and time since allo-HSCT||r = -0.044; p = 0.773 (NS)||r = -0.112; p = 0.463 (NS)|
The significance of differences was evaluated based on the Mann-Whitney U test **p < 0.01 a-a, ***p < 0.001; b-b, **p < 0.01 c-c, ***p < 0.001 d-d, *p < 0.05 e-e, *p < 0.05 f-f
Spearman’s correlation coefficient, NS – insignificant correlation
The mean resting salivary levels of lysozyme in post-transplant patients were comparable to those in patients in the control group (tab. 2). The mean stimulated salivary levels of lysozyme in post-transplant patients were statistically insignificantly higher compared to those in healthy individuals (tab. 2). It was noted that significant fluctuations and a large scatter in lysozyme levels occurred in post-allo-HSCT patients, particularly in Group I, whereas only minor differences in the same parameter were found in the control group. No statistically significant relationship was found in lysozyme levels and resting/stimulated salivary flow rate between post-allo-HSCT patients and controls (tab. 2). Mixed resting and stimulated salivary levels of lactoferrin were statistically significantly higher in the whole group of post-allo-HSCT patients compared to controls (p < 0.01 and p < 0.001) (tab.3). It should be noted that both resting and stimulated salivary levels of lactoferrin were particularly high in the earliest period (Group I) and the latest period (Group III) after transplantation, and differed statistically significantly from the mean levels in the control group. Lactoferrin levels increased statistically significantly with a decrease in the resting salivary flow rate in both post-allo-HSCT patients and controls (p < 0.05). For stimulated saliva, this relationship was found only in post-allo-HSCT patients (p < 0.05) (tab. 3).
In terms of oral cavity pathology in patients in the late period after allogeneic HSCT, the answer to the question whether there was a change in the levels of selected salivary parameters, representing salivary defence mechanisms, was interesting. Our findings indicate that there was no statistically significant difference in the mean resting or stimulated salivary levels of lysozyme between all post-allo-HSCT patients and the controls. It was noted that the levels of lysozyme in post-allo-HSCT patients were very varied and dispersed, in favour of higher values, particularly in patients in the first year after transplantation. It is a known fact that the oral environment is subjected to a variety of factors, which can potentially modulate salivary lysozyme levels. According to some authors, such effects are primarily caused by immunosuppressants (20). Pajari et al. (20) found significantly increased salivary levels of muramidase during chemotherapy in their study. On the contrary, Karolewska et al. showed no differences between resting salivary levels of lysozyme before and during therapy in their study in children treated for leukemia. However, a minor tendency of lysozyme levels to increase with the treatment progress was observed, which, according to the authors, was related to oral mycosis (21). The occurrence of some of the local and systemic diseases, inflammatory in particular, is believed to be of significant importance (22, 23). Rathnayake et al. found increased muramidase levels in the saliva from patients with periodontitis (23). Similar changes were observed in patients with ulcerative colitis, musculoskeletal and cardiovascular diseases and diabetes (22). Our study showed that more than 70% of patients developed the symptoms of oral mucosa inflammation. The symptoms occurred particularly in patients in the first year after allogeneic HSCT. Inflammatory lesions as well as the coexistent systemic diseases and their treatment could have accounted for the differences in the levels of salivary lysozyme. We have not found a statistically significant correlation between resting and stimulated salivary flow rate and lysozyme levels. Such a correlation was also absent in the studies presented by Zipp et al., who assessed stimulated salivary flow rate in patients with Sjögren’s syndrome (24). Zalewska et al. found in their study in 30 patients with rheumatoid arthritis that patients with reduced secretion and, at the same time, lower lysozyme secretion compared to patients with unimpaired salivary secretion, had high salivary levels of lysozyme. The authors postulate that the increase in the total lysozyme levels may be related to salivary gland infiltration by inflammatory cells and their release of proteins. The simultaneous reduction in the release of lysozyme may result from glandular impairment due to pathological processes, such as fibrosis or atrophy (25). It should be noted that similar changes in the glandular function are also often observed in patients after allogeneic HSCT with graft-versus-host disease (26).
The analysis of resting and stimulated salivary levels of lactoferrin revealed that the levels were statistically significantly higher in post-allo-HSCT patients compared to controls. The mean lactoferrin levels in post-transplant patients were comparable to those observed by Eliasson et al. in patients with Sjögren’s syndrome (27). Imanguli et al., who analysed saliva in patients 6 weeks after allogeneic HSCT, found numerous changes in the protein profile compared to the pre-transplantation period, including e.g. significantly increased lactoferrin levels (28). Our findings indicate that lactoferrin levels were statistically significantly correlated with both resting and stimulated salivary flow rate (negative correlation). A similar relationship was noted by Sikorska et al., who assessed salivary lactoferrin levels in healthy individuals (17). The analysis of literature and own findings suggests that the increased levels of lactoferrin in post- allo-HSCT patients could be due to e.g. gingival inflammation. It was found that up to 56.8% of post-transplant patients participating in our study had gingival inflammation of varying degrees (data not included in the table). Almståhl et al., who assessed oral health in patients with Sjögren’s syndrome, also suggested marginal periodontitis as a possible reason for the increased salivary lactoferrin levels in these patients (8). Dodds et al. believe that high salivary levels of lactoferrin may be due to salivary gland inflammation (6). According to other authors, increased salivary levels of lactoferrin are caused by altered permeability of the oral mucosa to plasma components, which is found in patients with chronic reduction in salivary flow (27). Such a pathomechanism could be also found in our patients as up to 39 out of 45 subjects developed clinical symptoms indicating salivary deficiency, as shown in previous publications (4). Based on the results, it was found that the time elapsed since allogeneic HSCT had no decisive influence on lactoferrin levels. Izutsu et al., who investigated post-transplant patients, found a gradual increase in salivary levels of this glycoprotein over time after transplantation (26).
No differences were found in resting and stimulated salivary levels between post-allo-HSCT patients and healthy individuals. Significant fluctuations and scatter in the levels of lysozyme in both resting and stimulated saliva were noticeable in post-allo-HSCT patients. Individuals with very high mixed salivary lysozyme could be identified in each of the three groups formed based on the time elapsed since transplantation. Mixed resting and stimulated salivary levels of lactoferrin were very high and statistically significantly higher in post-allo-HSCT patients compared to healthy individuals. Mixed resting and stimulated salivary levels of lysozyme and lactoferrin did not depend statistically significantly on the time elapsed since transplantation. The levels of lactoferrin increased statistically significantly with a decrease in resting and stimulated salivary flow rate.
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