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Immunostimulating and protective effects of an oral polybacterial immunomodulator ‘Dentavax’ in a rabbit experimental model
International Journal of Immunopharmacology 22 (2000) 843 – 854 www.elsevier.com/locate/ijimmpharm
Immunostimulating and protective effects of an oral polybacterial immunomodulator ‘Dentavax’ in a rabbit experimental model Snejina Marinova a,*, Ljubka Tchorbadjiiska b, Bogdan Petrunov a, Jordan Cvetanov a,w, Plamen Nenkov a, Dora Konstantinova a, Roumiana Markova a a
Head Laboratory of Humoral and Mucosal Immunity, National Center of Infectious and Parasitic Diseases, 26 Yanko Sakazo6 Bl6d, Sofia 1504, Bulgaria b Higher Medical School, 5 St G. Sofiisky St., Sofia 1606, Bulgaria Received 6 January 2000; received in revised form 24 March 2000; accepted 1 June 2000
Abstract The immunostimulating and protective effects of an oral polybacterial immunomodulator, Dentavax (D), composed of killed cells from Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Candida albicans and Lactobacillus acidophilus and their lysates, have been investigated on an experimental rabbit model. In this model, mixed suspensions of the above bacterial wild strains have been injected in six sides of oral mucosa. A long-lasting inflammation with the development of infiltrates and confluating abscesses has been observed. The influence of orally given Dentavax on the course of the model infection as well as on the dynamics of the immune response has been studied. A two-fold decrease in the duration and severity of inflammatory reaction, confirmed by the histological findings, has been registered. In immunised animals, an activation of polymorphonuclear phagocytosis, together with stimulation of humoral systemic and mucosal immunity with synthesis of specific serum (predominantly, IgG) and coproantibodies (predominantly, S-IgA) determined by ELISA, has been found. The results obtained proved the strong immunostimulating and protective effects of the preparation D, which is meant for the prophylaxis and treatment of inflammatory periodontal diseases. © 2000 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. Keywords: Oral immunomodulator; Polybacterial immunomodulator; Dentavax; Experimental model; Model infection; Oral inflammations; Periodontal disease; Mucosal immunity; S-IgA; Protection
* Corresponding author. Tel.: + 359-2-4347304; fax: + 3592-9433075. E-mail address: [email protected] (S. Marinova). w Deceased.
1. Introduction Oral mucosa normally prevents the penetration of micro-organisms and macromolecules that
0192-0561/00/$20.00 © 2000 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. PII: S0192-0561(00)00044-8
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might be antigenic. The mouth is part of the mucosal lining of the body and structurally shows similarities with tissues of gut and lungs [5,6]. Superficial periodontal tissues are constantly exposed to a mixed microbial flora, which can elicit inflammations and tissue destruction [40]. Periodontal disease is the general description given to the inflammatory response of oral mucosa and connective tissues to the bacterial accumulation on the teeth [24]. These diseases are widely spread infections in humans and are a great problem in medical practice. Numerous longitudinal studies demonstrate that the strategy of periodontal therapy is not consistent [9,13,15,16]. Despite the increased knowledge of the etiology and pathogenesis of periodontal infections, diagnostic markers for predicting the progression of and the response to treatment are still limited in a clinical setting [32]. A great number of oral bacterial species are polyresistent to antibiotics and chemotherapeutics. This fact focuses the attention on new approaches to prevention and control of the disease, such as vaccines and immunomodulators [7,18,19,33,34,36]. In a previous paper [26], we describe the polybacterial immunomodulator Dentavax (D), for the prophylaxis and treatment of inflammations of oral mucosa and parodont, constructed by our team in the National Center of Infectious and Parasitic Diseases, Sofia. The preparation has been tested on a guinea pig model, in which after the oral immunisation of animals, the specific and non-specific lymphoproliferative responses of T and B cells have been determined in peripheral blood, spleen, mesenteric lymph nodes and Peyer’s patches. The results obtained from the lymphoproliferative and the electron-microscopic studies showed that after the oral application of Dentavax an adequate immune response and a potent induction of local and systemic immunity have been stimulated. In the present paper, data for the immunostimulating and protective effects of the preparation on an experimental rabbit model are presented.
2. Materials and methods
2.1. Animals Chinchilla rabbits (3500–3800 g) conventionally housed were used throughout the study.
2.2. Selection of bacterial species included in the preparation of denta6ax In the Laboratory of Microbiology at the Faculty of Stomatology in Sofia, an investigation on 240 specimens from patients with inflammatory diseases of oral mucosa and the facio-maxillary region for a period of 3 years has been made. According to the incidence of various bacterial species found, the following were chosen for the construction of the polybacterial preparation — Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Candida albicans and Lactobacillus acidophilus. The identification of bacterial strains included in the preparation has been previously described [26]. Thus, the created immunomodulator Dentavax consists of killed bacterial cells and their lysates of the five bacterial species described above [36]. For this reason, the selected strains have been cultivated separately in a bioreactor. Bacterial density has been estimated by spectrophotometry and standardisation has been carried out. Bacterial cultures have been killed so that a part of the cells were lysed. Each dose of Dentavax is of 35 mg.
2.3. Experimental model of inflammatory diseases of oral mucosa and parodont An experimental rabbit model, created by Bulgarian authors [41], in which the influence of helio-neon laser on experimental inflammatory diseases of oral cavity and parodont with bacterial suspensions from S. pyogenes, S. epidermidis, N. sicca, C. xerosis and C. albicans has been successfully used in our investigation. At the same time, in our experimental model, a new bacterial association from the five bacterial species described has been included.
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Bacterial suspensions in apyrogenic saline have been prepared with each of the five wild bacterial species. A standardisation of the concentration of each suspension has been made in accordance with the experimental settings after which all suspensions were mixed in equal volumes. Two hundred microlitres of the mixture was injected with a tuberculin syringe at six sites in the mouth — two sites in the right and in the left part of the buccal mucosa of the maxilla and mandible regions and two sites, up and down, in the interdental papillae. Three groups of animals, each of eight, have been included in the experiment. First group: rabbits were immunised for 10 consecutive days three times daily with a dose of 35 mg Dentavax per day and challenged (according to the above model) after the application of the last dose; Second group: rabbits were immunised according to the same schedule, but were challenged after a 10-day period; Third group: nonimmunised animals, infected only.
2.4. Immunisation procedures Oral immunisation of rabbits has been carried out before challenge according to the following schedule. Animals received orally three times daily 105 mg of the preparation (each dose of 35 mg diluted in 1 ml saline) for 10 consecutive days. The control group received only 1 ml saline three times a day. In order to study the dynamics of primary and secondary immune responses 5 months after the last day of the first immunisation, a re-immunisation has been done only in groups of animals that were not challenged.
2.5. Materials tested Peripheral blood was collected from the ear’s marginal vein of rabbits. Inactivated sera at 56°C for 30 min was stored at −20°C. Fresh faeces were collected in saline containing a protease inhibitor Contrical (VEB Arzneimittelwerk, Dresden), 100 IU/ml. The proportion, faeces/saline, was 1:10. Then faeces were suspended, mixed on Vortex and
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centrifuged at 15 000 rpm for 15 min. The supernatant was inactivated at 56°C for 30 min and stored at − 20°C.
2.6. Phagocytic acti6ity of peripheral blood (PB) polymorphonuclears (PMNs) The method of Vymola [42] has been used. In brief, killed cells of Saccharomyces cere6isiae have been used for phagocytosis, i.e. 200 ml of 3 ×108/ml bacterial cells were added to 2 ml of heparinized blood (10 U/ml). After an incubation period of 1 h at 37°C, in the presence of 5% CO2, smears have been prepared on glass slides, fixed in absolute methanol (Sigma) and stained with 10% Giemsa (Sigma). The phagocytic activity of PMNs was examined under a light microscope (× 400) and was estimated on the basis of the phagocytic index and the phagocytic number.
2.7. Determination of specific antibodies in serum and faecal samples Specific antibodies were measured in ELISA microplates (Greiner Labortechnik, Austria). The procedure was as follows. Killed bacterial cells from the five species composing Dentavax were washed three times in saline and resuspended in a concentration of 108 cells per ml in a chloroform– alcohol solution, in the proportion 1:10, using a method applied for LPS antigens [11]. Different bacterial suspensions were distributed in microplates and fixed for 24 h at room temperature, and washed four times with phosphate buffered saline (PBS) with pH 7.2, containing 0.05% Tween 20 (Merck) and blocked for 90 min at 37°C with 2% human serum albumin (Sigma) diluted in PBS. The plates were washed again and diluted sera or faecal extracts were added, incubated for 120 min and washed. Peroxydase conjugates of goat antirabbit S-IgA and IgG sera (NCIPD, Bulgaria), in previously determined working dilutions, were incubated at 37°C for 90 min. After the last wash, orthophenylen-diamine (Sigma) 0.4 mg/ml, diluted in phosphate–citrate buffer (pH 5.0), containing 0.01% hydrogen peroxide (Merck) was added for 30 min and stopped with 25 ml 4H2SO4. The optical density was estimated with a Multiscan MCC/340 (Labsystems) at 495 nm.
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2.8. Histological studies Specimens for histological studies have been taken always from one and the same locus, i.e. from the upper and lower lips and from the interdental gingiva. Materials have been fixed in 10% formalin, treated after a routine technique and imbedded in paraffin blocks. Cuts have been made on Reichert microtome and stained with hemalaun-eosine.
2.9. Statistical analysis The results have been statistically evaluated by Student’s t-test. A value of P B0.05 was considered significant. Each experiment was repeated three times.
3. Results In the process of creation of the rabbit model, several preliminary experiments have been carried out and showed that the five virulent bacterial cultures with concentrations of 102 – 106 cells per ml, mixed in equal proportions, did not produce an inflammatory reaction during the observation period of 1 month. Initial changes, mainly hyperaemia and oedema, were registered with a bacterial concentration of 107 cells per ml. The greatest manifestation of the inflammatory reaction was achieved at a dose of 1010 cells per ml, but some of the animals died, so the lower (5 ×109) dose has been chosen as the most suitable one. The reason for that choice was the survival of all animals and the development of 100% morbidity, expressed as intensive local inflammatory reactions. They were expressed as propagating infiltrates and abscesses around the lips, identical to those observed in the case of the dose with 1010 cells per ml. The protective effect of orally applied Dentavax has been studied in three groups, each of eight rabbits, i.e. two groups of immunised rabbits and one group of controls, in which a model infection has been produced with the optimal dose of 5× 109 cells per ml.
In nonimmunised animals, the initial symptoms appeared on day 4 and were manifested by hyperaemia and oedema at the site of application of the mixed bacterial suspension. In 50% of those animals, infiltrates of great induration and massive abscesses developed. Between day 7 and 10, the infiltrates enlarged with increased degree of induration. That was especially demonstrative in the lateral mucosal fields. In the interdental papillae of the upper and lower gums, the infiltrates propagated through the lips to the nose. In all animals, opened and closed abscesses of 6–10 mm in diameter were found and purulent material leaked from the interdental space to the teeth. Between day 10 and 20, the process intensified and very often skin and lateral labial abscesses were observed too. Incisions had to be done as the abscesses enlarged up to 15/15 mm. Between day 30 and 60, a significant decrease of all these manifestations was found. However, the infiltrates subsided slowly, as shown in Fig. 1. In both groups of immunised animals, hyperaemia and oedema were observed between day 5 and 7. In a small percentage of animals, initial infiltrates and small abscesses were found. Between day 10 and 15, infiltrates and abscesses were found in almost all immunised animals too. At that time, the pathological changes were less manifested in comparison to the described control group (nonimmunised animals) where the infiltrates were moderately expressed and disappeared quickly, as shown in Fig. 1. In some cases, purulent material leaked from the gingiva to the teeth, but only for a few days, after which a quick improvement of mucosa occurred. The disappearance of infiltrates in oral mucosa and parodont was comparatively slower. It is very encouraging that the period of complete recovery of both groups of immunised animals was significantly shorter than that in the control group (Table 1). These data strongly demonstrate the protective effect of the polybacterial immunomodulator Dentavax on the model infection investigated in this study. Histological examinations have been made in both immunised and non-immunised animals in the late period of infection as well as in healthy rabbits. In materials taken from skin areas of the
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upper lip of all animals tested, a great difference in the degree of recovery after the model infection in immunised and non-immunised rabbits has been observed (Fig. 2a, b and Fig. 3a, b). In nonimmunised animals, the process of recovery developed very slowly and the comparison with the histological data obtained after immunisation on day 23 showed a significant quantitative difference (Fig. 2a, b and Fig. 3a, b). In only infected animals the lymphoplasmocytic infiltrates included leukocytes too. At the same time an abnormal acanthosis in stratum spinosum has been observed. Our observations showed that oral immunisation of rabbits with Dentavax stimulated the
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phagocytic activity of PMNs. In only immunised animals, which received Dentavax for 10 consecutive days, an elevation of the phagocytic index and the phagocytic number was found on day 4 from the beginning of the immunisation. Peak values of these parameters were reached on day 2 after the last immunisation dose, after which a slow decrease has been found (Fig. 4). In immunised and infected animals (the model rabbit infection), a very good phagocytic activity has been achieved even on day 3 in both the groups studied. In the group of animals challenged immediately after immunisation, peak values of phagocytosis were registered on day 14 when the highest values of phagocytic index and
Fig. 1. Experimental rabbit model, 1.2 ml of mixed bacterial suspension from five bacterial species composing Dentavax (5 ×109 bacterial cells per ml each) was injected in the mouth mucosa of rabbits at six points as follows. Two areas of the left and right side of the maxilla and mandibula and two areas in the interdental papilae. The reaction of inflammation-infiltrates and abscesses were followed-up — I group, immunised for 10 consecutive days, three times daily with a dose of 35 mg D. The model infection was induced immediately after immunisation; II group, the model infection was induced 10 days post-immunisation; III group, animals infected only. Table 1 Effect of dentavax on recovery process in immunised, as compared to non-immunised rabbits after a model infection Groups
Day of complete recovery X(
I, immunised and infected immediately II, immunised and infected after a 10-day interval III, control-infected only
44.6 920.62 42 9 14.1 100 9 16.1
Minimum 67 57 126
Group comparison Maximum 23 23 90
I–III, PB0.05 II–III, PB0.001 I–II, PB0.05
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quantities and the typical secondary IgA immune response was absent. The oral application of Dentavax resulted in the stimulation of mucosal immunity (Figs. 7 and 8). Even on day 7, after the immunisation, an increase of the values of specific S-IgA coproantibodies was observed. Significant quantitative differences, approximately twice higher, were found after re-immunisation (Fig. 7). Data from the measurement of specific IgG coproantibodies were analogous to those of S-IgA, but the secondary IgG response to some of the components of Dentavax was found smaller (Fig. 8). These data demonstrate the stimulation of specific local secretory immune response, although the faecal
Fig. 2. Experimental model of a non-immunised rabbit on the 23rd day after infection. Acanthosis in the epidermis; inflammatory infiltrates consisting of leukocytes, lymphocytes and plasmocytes in the dermal papillae, as well as perivascularly and defusely in derma; interdental gingiva (2a) and upper lip (2b); Hemalaun-eosin staining; Magnification, ×125.
phagocytic number were reached (Fig. 5). In the group challenged 10 days after immunisation, peak values were registered a week earlier, i.e. on day 7. Specific humoral immune responses to the selected bacterial species, which are components of D, were tested on rabbits that were orally immunised and re-immunised 5 months later. The data presented in Fig. 6 demonstrate a weak IgG response provoked by the immunisation. At all terms of examination after re-immunisation, specific serum antibodies reached higher levels. Specific IgA antibodies were in significantly lower
Fig. 3. Experimental model of an immunised and challenged immediately after immunisation with Dentavax rabbit. Rare lymphoplasmocytic infiltrates consisting of five to nine cells, situated in the derma of interdental gingiva (3a) and upper lip (3b); Hemalaun-eosine staining; Magnification, × 125.
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Fig. 4. Rabbits were orally immunised for 10 consecutive days, three times daily with a dose of 35 mg D. The phagocyte index and phagocitic number of peripheral blood PMNs were assessed on day 4 during immunisation (d.i.) and on days 2, 7, 10 and 21 after immunisation (a.i.).
Fig. 5. Rabbits were orally immunised for 10 consecutive days, three times daily with a dose of 35 mg D. A model infection was induced immediately after the immunisation. Phagocytic activity of peripheral blood PMNs was assessed on days 3, 7, 14 and 21 after challenge.
samples were significantly diluted (1:50). In our study, we have also found that the immunisation of rabbits with Dentavax long after the model infection (5 months, data not shown) stimulated typical secondary type-specific S-IgA responses. This fact suggested that Dentavax could produce mucosal immune responses after or during oral inflammatory diseases.
4. Discussion One of the basic problems in the investigation of inflammatory diseases of oral cavity is the creation of suitable experimental models. A number of attempts have been made to produce mucosal and periodontal disorders in different model animals, e.g. rabbits, rats, hamsters, ferrets
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[8,10,17,19,25,45], and experimental gingivitis in humans too [21 – 23] In rabbits, parenterally immunised, experiments have been done to obtain periodontal lesions after local application of various antigens [27]. Using actinomycetes, in hamsters and gnotobiotic rats, inflammatory processes have been developed [14]. Diabetes models in rats have been also used for induction of bone resorption. In those cases, the most often applied mi-
croorganism was Porphyromonas gingi6alis [18,19], the same one as in the experimental infection in rabbits [8]. A great difference was found in the morphology of teeth periodontal complexes, mouth microflora and the immune responses between the already described rodent models and humans [31]. Dogs and monkeys, especially chimpanzees, are appropriate models for studying chronic periodontal diseases because of the close
Fig. 6. Rabbits were orally immunised for 10 consecutive days, three times daily with a dose of 35 mg D, and re-immunised after a period of 5 months following the same schedule. Specific serum IgG antibodies were assessed on days 7, 14 and 21 after immunisation and re-immunisation.
Fig. 7. Rabbits were orally immunised for 10 consecutive days, three times daily with a dose of 35 mg D, and re-immunised after a period of 5 months following the same schedule. Specific S-IgA coproantibodies were assessed on days 7, 14 and 21 after immunisation and re-immunisation.
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Fig. 8. Rabbits were orally immunised for 10 consecutive days, three times daily with a dose of 35 mg D, and re-immunised after a period of 5 months following the same schedule. Specific IgG coproantibodies were assessed on days 7, 14 and 21 after immunisation and re-immunisation.
relationship with human microflora, but the resorption processes take a lot of time, even years [37]. At present, a model of inflammatory disease, analogous to that in humans has not been created. In the existing models, usually a single microorganism has been used to provoke pathological processes. The rabbit model described in the present work has the following advantages. First of all, pathological processes were induced by a mixed culture of bacterial species, the most common etiologic factors of oral inflammatory diseases in Bulgaria. Some authors indicate that demographic variables within a study population can influence factors associated with periodontal diseases and disease characteristics [23,38] Our model offers the opportunity to investigate the kinetics of the pathological process after a comparatively short period of time, as well as to register the protective effect of the preparation. At the same time, some of the immunological parameters could be studied dynamically. The protective effect of Dentavax in our model is demonstrated in Table 1 and Figs. 1 and 2a, b and Fig. 3a, b). It is very encouraging that the period of complete recuperation of all immunised animals was significantly shorter than that in the group of nonimmunised ones. The fact that there
was no significant difference in the duration of inflammation between the two immunised groups was a good proof that the protective effect of Dentavax did not depend on the 10-day interval between re-immunisation and challenge of animals. Oral mucosa is protected by both specific and non-specific defence mechanisms among which PMNs play a key role [6], as cells primarily responsive to microbial immunomodulators [4]. Several investigators have reported defective PMNs function in some patients [29,30], and in experimental periodontal lesions in animals [45]. Patients with neutrophil defects often suffer from oral mucosal ulcerations, gingivitis, and periodontitis [12]. In our study, data received after the oral application of Dentavax demonstrated the stimulating effect of the preparation on the phagocytic activity of PB PMNs for a long period of time as shown in Fig. 4. The activation of phagocytosis was most demonstrative in the course of the infectious process in rabbits challenged immediately after immunisation (Fig. 5). In a previous paper [39], we demonstrated analogous results, but on guinea pig PB PMNs. It is well known that evaluation of antibodies in faeces is very difficult as the gastrointestinal tract is an open system and a significant part of anti-
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bodies locally secreted by the intestinal mucosa is excreted with faeces. The data presented in Figs. 7 and 8 prove the stimulation of good specific mucosal immune responses in the gastrointestinal tract, especially after re-immunisation, which are demonstrated by the secretion of S-IgA predominantly, as well as IgG coproantibodies to all components of D. This fact demonstrates that, Dentavax induces strong mucosal immunity, protecting oral mucosa from inflammatory diseases. The protective effect of S-IgA has been studied intensively. There is evidence that secretory immunoglobulins inhibit the adherence of some of the most common bacterial species in the mouth [17,43]. The protection rendered by Dentavax in our experimental model allowed us to suggest that, specific S-IgA antibodies secreted in large amounts by oral mucosa influenced the dynamics of inflammatory reactions which subsided more easily and for a significantly shorter period of time. Our observations from the present study are in agreement with the general common mucosal immune system concept, according to which when an antigen stimulus is given at an inductive mucosal site, S-IgA responses are triggered in all other mucosal effector sides [1,28]. Recent data depict the priority role of the nasal-associated lymphoid tissue (NALT) [20] in the stimulation of mucosal immunity [2,44]. These facts are in support of our assumption for the effectiveness of Dentavax in the treatment of inflammatory diseases of oral mucosa and parodont in humans, because of its oral application which is the peroral one (buccal tablets for sucking). In this way, Dentavax acts as a strong antigenic stimulus in two of the most important sites of induction of mucosal immunity, NALT and Peyer’s patches [3,35]. At the same time, the preparation stimulates a good specific systemic humoral immune response with the production of IgG antibodies, predominantly (Fig. 6). In conclusion, the described experimental rabbit model of oral inflammation is a convenient one for the investigation of immunologic effectiveness of the newly created immunomodulator D. The main advantage of this model is the possibility to study the recovery of oral inflammation after treatment with Dentavax for a short
period of time as demonstrated by our histological findings. The dynamics of morphological changes in immunised and non-immunised animals confirm the positive effect of the preparation in the context of the nature and duration of the inflammatory reaction. The oral application of the immunomodulator stimulates a strong humoral systemic and local mucosal as well as cellular immune responses, demonstrated by the secretion of specific serum and coproantibodies and activation of polymorphonuclear phagocytosis. There is a compelling need for new ways of prophylaxis and treatment of inflammations of oral mucosa and parodont we propose, that in clinical practice, Dentavax will be a suitable pharmacologic agent.
Acknowledgements We are indebted to Dr Sasha Nikolaeva, Head Laboratory of Immunomorphology and Electronmicroscopy at the National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria, for kindly preparing the materials for the histological studies and their interpretation. This work was supported by a grant-in-aid for scientific research from the Bulgarian Ministry of Education and Science.
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[21] Lehner T, Wilson JMA, Challacombe JJ, Ivanyi L. Sequential cell-mediated immune responses in experimental gingivitis in man. Clin Exp Immunol 1974;16:481 – 92. [22] Lo¨e H, Theilade E, Jensen SB. Experimental gingivitis in man. J Periodontol 1965;36:177 – 87. [23] Lo¨e H, Brown LJ. Early onset periodontal disease in the United States of America. J Periodontol 1991;62:608–16. [24] Loesche WJ. Bacterial mediators in periodontal disease. Clin Infect Dis 1993;16(Suppl. 4):203 – 10. [25] Mac N, Lamon R, Thomas HF. J Periodontol 1993;64(4):285. [26] Markova R, Marinova S, Petrunov B, Cvetanov J, Nenkov P, Radinov A, Tchorbadjiiska L, Konstantinova D. Stimulating effect of an oral polybacterial immunomodulator on the proliferative activity of guinea pig lymphocytes. Int J Immunopharmacol 1997;19(4):205–14. [27] McDougall WA. The effect of topical antigen on the gingiva of sensitised rabbits. J Periodont Res 1974;9:153– 69. [28] Mestecky J, Abraham R, Ogra PL. Common mucosal immune system and strategies for development of vaccines effective at the mucosal surfaces. In: Ogra PL, Mestecky J, Lamm ME, Strober W, McGhee JR, Bienenstock J, editors. Handbook of Mucosal Immunology. San Diego: Academic Press, 1994. [29] Mouynet P, Picot C, Nicolas P, Genetet B, Apiou J, Genetet N, Michel JF. Ex vivo studies of polymorphonuclear neutrophils from patients with early-onset periodontitis. III. CR3 and LFA-1 expression by peripheral blood and gingival crevicular polymorphonuclear neutrophils. J Clin Periodontol 1995a;22:110 – 7. [30] Mouynet P, Genetet N, Picot C, Apiou J, Nicolas P, Michel JF. Expression of the ?-subunit of CR4 (CD 11c) by peripheral blood and crevicular fluid neutrophils. Eur J Oral Sci 1995b;103:413– 5. [31] Navia JM. Experimental periodontal disease. In: Animal Model in Dental Research. University Ala, 1977:312–33. [32] Nieminen A, Asikainen S, Torkko H, Kari K, Uitto VJ, Saxen L. Value of some laboratory and clinical measurements in the treatment plan for advanced periodontitis. J Clin Periodontol 1996;23:572 – 81. [33] Page RC. Vaccine development for periodontal diseases. Mucosal Immunol Update 1995;3(2):9 – 11. [34] Persson GR, Engel LD, Whitney CW, Weinbeg A, Moncla BJ, Darveau RP, Houston L, Braham P, Page RC. Macaca fascicularis as a model in which to assess the safety and efficacy of a vaccine for periodontitis. Oral Microbiol Immunol 1994;9:104 – 11. [35] Perry M, Whyte A. Immunology of the tonsils. Immunol Today 1998;19(9):414– 20. [36] Petrunov B, Nenkov P, Marinova S, Cvetanov J, Konstantinova D, Tchorbadjiiska L. A new peroral immunostimulator (polybacterial vaccine) for immunotherapy and immunoprophylaxis of nonspecific inflammations of oral mucosa and parodont-‘Dentavax’. Infectology 1993;XX(2):24– 31.
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[37] Slots J, Hausmann E. Longitudinal study of experimentally induced periodontal disease in Macaca arctoides; relationship between microflora and alveolar bone loss. Infect Immun 1979;23:260–7. [38] Schenkein HA, Burmeister JA, Koertge TE, Brooks CN, Best AM, Moore LVH, Moore WEC. The influence of race and gender on periodontal microflora. J Periodontol 1993;64(4):292–6. [39] Taskov H, Petrunov B, Nenkov P, Marinova S, Dimitrova E. The influence of the oral immunostimulator dentavax on the phagocytic activity of PMNC in the peripheral blood of guinea pigs. Probl Infect Paras Dis, Sofia 1995;XXII:24–5. [40] Tonetti MS, Imboden MA, Gerber L, Lang NP, Laissue J, Mueller C. Localized expression of mRNA for phagocyte-specific chemotactic cytokines in human periodontal infections. Infect Immun 1994;62(9):4005–14. [41] Uzunov Z, Tchorbadjiiska L, Videnov L, Atanasova L, Vasilev M. Experimental histomorphological investiga-
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tion on the influence of helio-neon laser on bacterial-induced inflammation of oral mucosa and parodont. J Stomatol 1991;2:13 – 7. F. Vymola, Avicenum zarovatnike Nakladtelstvi, 1983, p. 225. Widerstrom L, Bratthall D, Hamberg K. Immunoglobulin A antibodies to mutans streptococci in human saliva and serum comparing fresh and subcultivated strains and activity in repeated saliva samples. Oral Microbiol Immunol 1994;9:278 – 83. Wu Hong Y, Russell MW. Nasal lymphoid tissue, intranasal immunization and compartmentalization of the common mucosal immune system. Immunol Res 1997;16(2):187– 201. Yoshinari N, Kameyama Y, Aoyama Y, Nishiyama H, Nogushi T. Effect of long-term methotrexate induced neutropenia on experimental periodontal. J Periodont Res 1994;29:393 – 400.
Immunostimulating and protective effects of an oral polybacterial immunomodulator ‘Dentavax’ in a rabbit experimental model Snejina Marinova a,*, Ljubka Tchorbadjiiska b, Bogdan Petrunov a, Jordan Cvetanov a,w, Plamen Nenkov a, Dora Konstantinova a, Roumiana Markova a a
Head Laboratory of Humoral and Mucosal Immunity, National Center of Infectious and Parasitic Diseases, 26 Yanko Sakazo6 Bl6d, Sofia 1504, Bulgaria b Higher Medical School, 5 St G. Sofiisky St., Sofia 1606, Bulgaria Received 6 January 2000; received in revised form 24 March 2000; accepted 1 June 2000
Abstract The immunostimulating and protective effects of an oral polybacterial immunomodulator, Dentavax (D), composed of killed cells from Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Candida albicans and Lactobacillus acidophilus and their lysates, have been investigated on an experimental rabbit model. In this model, mixed suspensions of the above bacterial wild strains have been injected in six sides of oral mucosa. A long-lasting inflammation with the development of infiltrates and confluating abscesses has been observed. The influence of orally given Dentavax on the course of the model infection as well as on the dynamics of the immune response has been studied. A two-fold decrease in the duration and severity of inflammatory reaction, confirmed by the histological findings, has been registered. In immunised animals, an activation of polymorphonuclear phagocytosis, together with stimulation of humoral systemic and mucosal immunity with synthesis of specific serum (predominantly, IgG) and coproantibodies (predominantly, S-IgA) determined by ELISA, has been found. The results obtained proved the strong immunostimulating and protective effects of the preparation D, which is meant for the prophylaxis and treatment of inflammatory periodontal diseases. © 2000 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. Keywords: Oral immunomodulator; Polybacterial immunomodulator; Dentavax; Experimental model; Model infection; Oral inflammations; Periodontal disease; Mucosal immunity; S-IgA; Protection
* Corresponding author. Tel.: + 359-2-4347304; fax: + 3592-9433075. E-mail address: [email protected] (S. Marinova). w Deceased.
1. Introduction Oral mucosa normally prevents the penetration of micro-organisms and macromolecules that
0192-0561/00/$20.00 © 2000 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. PII: S0192-0561(00)00044-8
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might be antigenic. The mouth is part of the mucosal lining of the body and structurally shows similarities with tissues of gut and lungs [5,6]. Superficial periodontal tissues are constantly exposed to a mixed microbial flora, which can elicit inflammations and tissue destruction [40]. Periodontal disease is the general description given to the inflammatory response of oral mucosa and connective tissues to the bacterial accumulation on the teeth [24]. These diseases are widely spread infections in humans and are a great problem in medical practice. Numerous longitudinal studies demonstrate that the strategy of periodontal therapy is not consistent [9,13,15,16]. Despite the increased knowledge of the etiology and pathogenesis of periodontal infections, diagnostic markers for predicting the progression of and the response to treatment are still limited in a clinical setting [32]. A great number of oral bacterial species are polyresistent to antibiotics and chemotherapeutics. This fact focuses the attention on new approaches to prevention and control of the disease, such as vaccines and immunomodulators [7,18,19,33,34,36]. In a previous paper [26], we describe the polybacterial immunomodulator Dentavax (D), for the prophylaxis and treatment of inflammations of oral mucosa and parodont, constructed by our team in the National Center of Infectious and Parasitic Diseases, Sofia. The preparation has been tested on a guinea pig model, in which after the oral immunisation of animals, the specific and non-specific lymphoproliferative responses of T and B cells have been determined in peripheral blood, spleen, mesenteric lymph nodes and Peyer’s patches. The results obtained from the lymphoproliferative and the electron-microscopic studies showed that after the oral application of Dentavax an adequate immune response and a potent induction of local and systemic immunity have been stimulated. In the present paper, data for the immunostimulating and protective effects of the preparation on an experimental rabbit model are presented.
2. Materials and methods
2.1. Animals Chinchilla rabbits (3500–3800 g) conventionally housed were used throughout the study.
2.2. Selection of bacterial species included in the preparation of denta6ax In the Laboratory of Microbiology at the Faculty of Stomatology in Sofia, an investigation on 240 specimens from patients with inflammatory diseases of oral mucosa and the facio-maxillary region for a period of 3 years has been made. According to the incidence of various bacterial species found, the following were chosen for the construction of the polybacterial preparation — Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Candida albicans and Lactobacillus acidophilus. The identification of bacterial strains included in the preparation has been previously described [26]. Thus, the created immunomodulator Dentavax consists of killed bacterial cells and their lysates of the five bacterial species described above [36]. For this reason, the selected strains have been cultivated separately in a bioreactor. Bacterial density has been estimated by spectrophotometry and standardisation has been carried out. Bacterial cultures have been killed so that a part of the cells were lysed. Each dose of Dentavax is of 35 mg.
2.3. Experimental model of inflammatory diseases of oral mucosa and parodont An experimental rabbit model, created by Bulgarian authors [41], in which the influence of helio-neon laser on experimental inflammatory diseases of oral cavity and parodont with bacterial suspensions from S. pyogenes, S. epidermidis, N. sicca, C. xerosis and C. albicans has been successfully used in our investigation. At the same time, in our experimental model, a new bacterial association from the five bacterial species described has been included.
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Bacterial suspensions in apyrogenic saline have been prepared with each of the five wild bacterial species. A standardisation of the concentration of each suspension has been made in accordance with the experimental settings after which all suspensions were mixed in equal volumes. Two hundred microlitres of the mixture was injected with a tuberculin syringe at six sites in the mouth — two sites in the right and in the left part of the buccal mucosa of the maxilla and mandible regions and two sites, up and down, in the interdental papillae. Three groups of animals, each of eight, have been included in the experiment. First group: rabbits were immunised for 10 consecutive days three times daily with a dose of 35 mg Dentavax per day and challenged (according to the above model) after the application of the last dose; Second group: rabbits were immunised according to the same schedule, but were challenged after a 10-day period; Third group: nonimmunised animals, infected only.
2.4. Immunisation procedures Oral immunisation of rabbits has been carried out before challenge according to the following schedule. Animals received orally three times daily 105 mg of the preparation (each dose of 35 mg diluted in 1 ml saline) for 10 consecutive days. The control group received only 1 ml saline three times a day. In order to study the dynamics of primary and secondary immune responses 5 months after the last day of the first immunisation, a re-immunisation has been done only in groups of animals that were not challenged.
2.5. Materials tested Peripheral blood was collected from the ear’s marginal vein of rabbits. Inactivated sera at 56°C for 30 min was stored at −20°C. Fresh faeces were collected in saline containing a protease inhibitor Contrical (VEB Arzneimittelwerk, Dresden), 100 IU/ml. The proportion, faeces/saline, was 1:10. Then faeces were suspended, mixed on Vortex and
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centrifuged at 15 000 rpm for 15 min. The supernatant was inactivated at 56°C for 30 min and stored at − 20°C.
2.6. Phagocytic acti6ity of peripheral blood (PB) polymorphonuclears (PMNs) The method of Vymola [42] has been used. In brief, killed cells of Saccharomyces cere6isiae have been used for phagocytosis, i.e. 200 ml of 3 ×108/ml bacterial cells were added to 2 ml of heparinized blood (10 U/ml). After an incubation period of 1 h at 37°C, in the presence of 5% CO2, smears have been prepared on glass slides, fixed in absolute methanol (Sigma) and stained with 10% Giemsa (Sigma). The phagocytic activity of PMNs was examined under a light microscope (× 400) and was estimated on the basis of the phagocytic index and the phagocytic number.
2.7. Determination of specific antibodies in serum and faecal samples Specific antibodies were measured in ELISA microplates (Greiner Labortechnik, Austria). The procedure was as follows. Killed bacterial cells from the five species composing Dentavax were washed three times in saline and resuspended in a concentration of 108 cells per ml in a chloroform– alcohol solution, in the proportion 1:10, using a method applied for LPS antigens [11]. Different bacterial suspensions were distributed in microplates and fixed for 24 h at room temperature, and washed four times with phosphate buffered saline (PBS) with pH 7.2, containing 0.05% Tween 20 (Merck) and blocked for 90 min at 37°C with 2% human serum albumin (Sigma) diluted in PBS. The plates were washed again and diluted sera or faecal extracts were added, incubated for 120 min and washed. Peroxydase conjugates of goat antirabbit S-IgA and IgG sera (NCIPD, Bulgaria), in previously determined working dilutions, were incubated at 37°C for 90 min. After the last wash, orthophenylen-diamine (Sigma) 0.4 mg/ml, diluted in phosphate–citrate buffer (pH 5.0), containing 0.01% hydrogen peroxide (Merck) was added for 30 min and stopped with 25 ml 4H2SO4. The optical density was estimated with a Multiscan MCC/340 (Labsystems) at 495 nm.
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2.8. Histological studies Specimens for histological studies have been taken always from one and the same locus, i.e. from the upper and lower lips and from the interdental gingiva. Materials have been fixed in 10% formalin, treated after a routine technique and imbedded in paraffin blocks. Cuts have been made on Reichert microtome and stained with hemalaun-eosine.
2.9. Statistical analysis The results have been statistically evaluated by Student’s t-test. A value of P B0.05 was considered significant. Each experiment was repeated three times.
3. Results In the process of creation of the rabbit model, several preliminary experiments have been carried out and showed that the five virulent bacterial cultures with concentrations of 102 – 106 cells per ml, mixed in equal proportions, did not produce an inflammatory reaction during the observation period of 1 month. Initial changes, mainly hyperaemia and oedema, were registered with a bacterial concentration of 107 cells per ml. The greatest manifestation of the inflammatory reaction was achieved at a dose of 1010 cells per ml, but some of the animals died, so the lower (5 ×109) dose has been chosen as the most suitable one. The reason for that choice was the survival of all animals and the development of 100% morbidity, expressed as intensive local inflammatory reactions. They were expressed as propagating infiltrates and abscesses around the lips, identical to those observed in the case of the dose with 1010 cells per ml. The protective effect of orally applied Dentavax has been studied in three groups, each of eight rabbits, i.e. two groups of immunised rabbits and one group of controls, in which a model infection has been produced with the optimal dose of 5× 109 cells per ml.
In nonimmunised animals, the initial symptoms appeared on day 4 and were manifested by hyperaemia and oedema at the site of application of the mixed bacterial suspension. In 50% of those animals, infiltrates of great induration and massive abscesses developed. Between day 7 and 10, the infiltrates enlarged with increased degree of induration. That was especially demonstrative in the lateral mucosal fields. In the interdental papillae of the upper and lower gums, the infiltrates propagated through the lips to the nose. In all animals, opened and closed abscesses of 6–10 mm in diameter were found and purulent material leaked from the interdental space to the teeth. Between day 10 and 20, the process intensified and very often skin and lateral labial abscesses were observed too. Incisions had to be done as the abscesses enlarged up to 15/15 mm. Between day 30 and 60, a significant decrease of all these manifestations was found. However, the infiltrates subsided slowly, as shown in Fig. 1. In both groups of immunised animals, hyperaemia and oedema were observed between day 5 and 7. In a small percentage of animals, initial infiltrates and small abscesses were found. Between day 10 and 15, infiltrates and abscesses were found in almost all immunised animals too. At that time, the pathological changes were less manifested in comparison to the described control group (nonimmunised animals) where the infiltrates were moderately expressed and disappeared quickly, as shown in Fig. 1. In some cases, purulent material leaked from the gingiva to the teeth, but only for a few days, after which a quick improvement of mucosa occurred. The disappearance of infiltrates in oral mucosa and parodont was comparatively slower. It is very encouraging that the period of complete recovery of both groups of immunised animals was significantly shorter than that in the control group (Table 1). These data strongly demonstrate the protective effect of the polybacterial immunomodulator Dentavax on the model infection investigated in this study. Histological examinations have been made in both immunised and non-immunised animals in the late period of infection as well as in healthy rabbits. In materials taken from skin areas of the
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upper lip of all animals tested, a great difference in the degree of recovery after the model infection in immunised and non-immunised rabbits has been observed (Fig. 2a, b and Fig. 3a, b). In nonimmunised animals, the process of recovery developed very slowly and the comparison with the histological data obtained after immunisation on day 23 showed a significant quantitative difference (Fig. 2a, b and Fig. 3a, b). In only infected animals the lymphoplasmocytic infiltrates included leukocytes too. At the same time an abnormal acanthosis in stratum spinosum has been observed. Our observations showed that oral immunisation of rabbits with Dentavax stimulated the
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phagocytic activity of PMNs. In only immunised animals, which received Dentavax for 10 consecutive days, an elevation of the phagocytic index and the phagocytic number was found on day 4 from the beginning of the immunisation. Peak values of these parameters were reached on day 2 after the last immunisation dose, after which a slow decrease has been found (Fig. 4). In immunised and infected animals (the model rabbit infection), a very good phagocytic activity has been achieved even on day 3 in both the groups studied. In the group of animals challenged immediately after immunisation, peak values of phagocytosis were registered on day 14 when the highest values of phagocytic index and
Fig. 1. Experimental rabbit model, 1.2 ml of mixed bacterial suspension from five bacterial species composing Dentavax (5 ×109 bacterial cells per ml each) was injected in the mouth mucosa of rabbits at six points as follows. Two areas of the left and right side of the maxilla and mandibula and two areas in the interdental papilae. The reaction of inflammation-infiltrates and abscesses were followed-up — I group, immunised for 10 consecutive days, three times daily with a dose of 35 mg D. The model infection was induced immediately after immunisation; II group, the model infection was induced 10 days post-immunisation; III group, animals infected only. Table 1 Effect of dentavax on recovery process in immunised, as compared to non-immunised rabbits after a model infection Groups
Day of complete recovery X(
I, immunised and infected immediately II, immunised and infected after a 10-day interval III, control-infected only
44.6 920.62 42 9 14.1 100 9 16.1
Minimum 67 57 126
Group comparison Maximum 23 23 90
I–III, PB0.05 II–III, PB0.001 I–II, PB0.05
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quantities and the typical secondary IgA immune response was absent. The oral application of Dentavax resulted in the stimulation of mucosal immunity (Figs. 7 and 8). Even on day 7, after the immunisation, an increase of the values of specific S-IgA coproantibodies was observed. Significant quantitative differences, approximately twice higher, were found after re-immunisation (Fig. 7). Data from the measurement of specific IgG coproantibodies were analogous to those of S-IgA, but the secondary IgG response to some of the components of Dentavax was found smaller (Fig. 8). These data demonstrate the stimulation of specific local secretory immune response, although the faecal
Fig. 2. Experimental model of a non-immunised rabbit on the 23rd day after infection. Acanthosis in the epidermis; inflammatory infiltrates consisting of leukocytes, lymphocytes and plasmocytes in the dermal papillae, as well as perivascularly and defusely in derma; interdental gingiva (2a) and upper lip (2b); Hemalaun-eosin staining; Magnification, ×125.
phagocytic number were reached (Fig. 5). In the group challenged 10 days after immunisation, peak values were registered a week earlier, i.e. on day 7. Specific humoral immune responses to the selected bacterial species, which are components of D, were tested on rabbits that were orally immunised and re-immunised 5 months later. The data presented in Fig. 6 demonstrate a weak IgG response provoked by the immunisation. At all terms of examination after re-immunisation, specific serum antibodies reached higher levels. Specific IgA antibodies were in significantly lower
Fig. 3. Experimental model of an immunised and challenged immediately after immunisation with Dentavax rabbit. Rare lymphoplasmocytic infiltrates consisting of five to nine cells, situated in the derma of interdental gingiva (3a) and upper lip (3b); Hemalaun-eosine staining; Magnification, × 125.
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Fig. 4. Rabbits were orally immunised for 10 consecutive days, three times daily with a dose of 35 mg D. The phagocyte index and phagocitic number of peripheral blood PMNs were assessed on day 4 during immunisation (d.i.) and on days 2, 7, 10 and 21 after immunisation (a.i.).
Fig. 5. Rabbits were orally immunised for 10 consecutive days, three times daily with a dose of 35 mg D. A model infection was induced immediately after the immunisation. Phagocytic activity of peripheral blood PMNs was assessed on days 3, 7, 14 and 21 after challenge.
samples were significantly diluted (1:50). In our study, we have also found that the immunisation of rabbits with Dentavax long after the model infection (5 months, data not shown) stimulated typical secondary type-specific S-IgA responses. This fact suggested that Dentavax could produce mucosal immune responses after or during oral inflammatory diseases.
4. Discussion One of the basic problems in the investigation of inflammatory diseases of oral cavity is the creation of suitable experimental models. A number of attempts have been made to produce mucosal and periodontal disorders in different model animals, e.g. rabbits, rats, hamsters, ferrets
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[8,10,17,19,25,45], and experimental gingivitis in humans too [21 – 23] In rabbits, parenterally immunised, experiments have been done to obtain periodontal lesions after local application of various antigens [27]. Using actinomycetes, in hamsters and gnotobiotic rats, inflammatory processes have been developed [14]. Diabetes models in rats have been also used for induction of bone resorption. In those cases, the most often applied mi-
croorganism was Porphyromonas gingi6alis [18,19], the same one as in the experimental infection in rabbits [8]. A great difference was found in the morphology of teeth periodontal complexes, mouth microflora and the immune responses between the already described rodent models and humans [31]. Dogs and monkeys, especially chimpanzees, are appropriate models for studying chronic periodontal diseases because of the close
Fig. 6. Rabbits were orally immunised for 10 consecutive days, three times daily with a dose of 35 mg D, and re-immunised after a period of 5 months following the same schedule. Specific serum IgG antibodies were assessed on days 7, 14 and 21 after immunisation and re-immunisation.
Fig. 7. Rabbits were orally immunised for 10 consecutive days, three times daily with a dose of 35 mg D, and re-immunised after a period of 5 months following the same schedule. Specific S-IgA coproantibodies were assessed on days 7, 14 and 21 after immunisation and re-immunisation.
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Fig. 8. Rabbits were orally immunised for 10 consecutive days, three times daily with a dose of 35 mg D, and re-immunised after a period of 5 months following the same schedule. Specific IgG coproantibodies were assessed on days 7, 14 and 21 after immunisation and re-immunisation.
relationship with human microflora, but the resorption processes take a lot of time, even years [37]. At present, a model of inflammatory disease, analogous to that in humans has not been created. In the existing models, usually a single microorganism has been used to provoke pathological processes. The rabbit model described in the present work has the following advantages. First of all, pathological processes were induced by a mixed culture of bacterial species, the most common etiologic factors of oral inflammatory diseases in Bulgaria. Some authors indicate that demographic variables within a study population can influence factors associated with periodontal diseases and disease characteristics [23,38] Our model offers the opportunity to investigate the kinetics of the pathological process after a comparatively short period of time, as well as to register the protective effect of the preparation. At the same time, some of the immunological parameters could be studied dynamically. The protective effect of Dentavax in our model is demonstrated in Table 1 and Figs. 1 and 2a, b and Fig. 3a, b). It is very encouraging that the period of complete recuperation of all immunised animals was significantly shorter than that in the group of nonimmunised ones. The fact that there
was no significant difference in the duration of inflammation between the two immunised groups was a good proof that the protective effect of Dentavax did not depend on the 10-day interval between re-immunisation and challenge of animals. Oral mucosa is protected by both specific and non-specific defence mechanisms among which PMNs play a key role [6], as cells primarily responsive to microbial immunomodulators [4]. Several investigators have reported defective PMNs function in some patients [29,30], and in experimental periodontal lesions in animals [45]. Patients with neutrophil defects often suffer from oral mucosal ulcerations, gingivitis, and periodontitis [12]. In our study, data received after the oral application of Dentavax demonstrated the stimulating effect of the preparation on the phagocytic activity of PB PMNs for a long period of time as shown in Fig. 4. The activation of phagocytosis was most demonstrative in the course of the infectious process in rabbits challenged immediately after immunisation (Fig. 5). In a previous paper [39], we demonstrated analogous results, but on guinea pig PB PMNs. It is well known that evaluation of antibodies in faeces is very difficult as the gastrointestinal tract is an open system and a significant part of anti-
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bodies locally secreted by the intestinal mucosa is excreted with faeces. The data presented in Figs. 7 and 8 prove the stimulation of good specific mucosal immune responses in the gastrointestinal tract, especially after re-immunisation, which are demonstrated by the secretion of S-IgA predominantly, as well as IgG coproantibodies to all components of D. This fact demonstrates that, Dentavax induces strong mucosal immunity, protecting oral mucosa from inflammatory diseases. The protective effect of S-IgA has been studied intensively. There is evidence that secretory immunoglobulins inhibit the adherence of some of the most common bacterial species in the mouth [17,43]. The protection rendered by Dentavax in our experimental model allowed us to suggest that, specific S-IgA antibodies secreted in large amounts by oral mucosa influenced the dynamics of inflammatory reactions which subsided more easily and for a significantly shorter period of time. Our observations from the present study are in agreement with the general common mucosal immune system concept, according to which when an antigen stimulus is given at an inductive mucosal site, S-IgA responses are triggered in all other mucosal effector sides [1,28]. Recent data depict the priority role of the nasal-associated lymphoid tissue (NALT) [20] in the stimulation of mucosal immunity [2,44]. These facts are in support of our assumption for the effectiveness of Dentavax in the treatment of inflammatory diseases of oral mucosa and parodont in humans, because of its oral application which is the peroral one (buccal tablets for sucking). In this way, Dentavax acts as a strong antigenic stimulus in two of the most important sites of induction of mucosal immunity, NALT and Peyer’s patches [3,35]. At the same time, the preparation stimulates a good specific systemic humoral immune response with the production of IgG antibodies, predominantly (Fig. 6). In conclusion, the described experimental rabbit model of oral inflammation is a convenient one for the investigation of immunologic effectiveness of the newly created immunomodulator D. The main advantage of this model is the possibility to study the recovery of oral inflammation after treatment with Dentavax for a short
period of time as demonstrated by our histological findings. The dynamics of morphological changes in immunised and non-immunised animals confirm the positive effect of the preparation in the context of the nature and duration of the inflammatory reaction. The oral application of the immunomodulator stimulates a strong humoral systemic and local mucosal as well as cellular immune responses, demonstrated by the secretion of specific serum and coproantibodies and activation of polymorphonuclear phagocytosis. There is a compelling need for new ways of prophylaxis and treatment of inflammations of oral mucosa and parodont we propose, that in clinical practice, Dentavax will be a suitable pharmacologic agent.
Acknowledgements We are indebted to Dr Sasha Nikolaeva, Head Laboratory of Immunomorphology and Electronmicroscopy at the National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria, for kindly preparing the materials for the histological studies and their interpretation. This work was supported by a grant-in-aid for scientific research from the Bulgarian Ministry of Education and Science.
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