The arginine-deiminase enzymatic system on gingivitis: preliminary pediatric study

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(1)Introduction. ni. In. te. “Sapienza” University of Rome, Rome, Italy 1 Department of Oral Sciences, Paediatric Dentistry Unit, (A. Polimeni) 2 Department of Infectious and Tropical Diseases (V. Vullo). The lactic bacteria are Gram-positive microorganisms with coccus or stick shape, which share a number of physiological and biochemical properties. They include some species of lactobacilli, lattococchi, streptococci, pediococchi. Several experimental evidences suggest the possibility of using lactic acid bacteria as a potential preventive or therapeutic approaches, alternative or complementary protocols prevention or treatment currently followed in several pathological conditions (Offenbacher, 1993; Mombelli, 1998) Specific components of Lactobacilli seem to have an intense effect on the modulation of immune response, on the activation of the reticuloendothelial system and on the regulating production of cytokines and inflammatory response (Malin, 1996; Naidu, 1999). In this way it is very important to the study conducted by Ulisse (Ulisse, 2001) in which was found an inhibitory action of Lactobacillus brevis against iNOS, in rat’s peritoneal macrophages stimulated in vitro with IFN-γ and lipopolysaccharide (LPS). In the light of these results, in this work, we have proposed to assess the anti inflammatory effects of Lactobacillus brevis in case of marginal gingivitis. The initial pathogenic event determines in guest’s organism tissues the production and synthesis of cytokines (Bucci, 2006). The excessive production of cytokines driving in a non-specific way the production of nitric oxide (NO). NO2, gaseous free radical, is fleeting and in the presence of oxygen is rapidly metabolized by nitrate to nitrite (NO). The increase of NO can not be seen as specific for a clinical condition, but rather as a reflection of an immuno-inflammatory activated state, in which cytokines, together with other mediators, led to a stimulation of NO-synthase in tissues and fluids (Ramberg, 1994; Persson, 1990). It is clear that excessive levels of NO are harmful and dangerous for the tissues and promote the progression of the inflammatory phenomena (De Luca, 1999). The plaque bacteria that have arginina-deiminase enzyme are: Streptococcus sanguinis, Streptococcus milleri, Streptococcus rattus, Lactobacillus fermentum, Streptococcus faecalis, Streptococcus faecium, Eubacterium nodatum species. Some strains of Lactobacillus brevis species (CD2) are surprisingly rich in “arginina-deiminasi” enzymatic system. It is believed that the enzyme may play an important role in the ecology of the mouth probably protecting oral bacteria that are less tolerant against acid environment, by the pH decrease (4.0) caused by acidophilous bacteria glycolysis (Lindhe, 1998; Page, 1996; Polimeni, 1995). This defense is expressed through an increase in pH associated with the production of ammonia. The action pad done by ammonia can be positive not only for the same plaque bacteria, but also for tooth and tissues of the guest. This enzyme uses the same iNOS substrate, inhibiting its action, and pursuing anti-inflammatory effects (Chambers, 1991; Lamster, 1987; Wong, 1999; Mizuho, 1996). The goal of the study has been to identify the possible medical applications, both preventively and therapeutically, of the lactic acid bacteria strains selected on the basis of specific chemical, physical and/or biological characteristics. In particular, objectives of this work were:. rn a. Gaetano Ierardo, DDS, PhD1 Maurizio Bossù, DDS, PhD1 Desirée Tarantino, DDS1 Vincenzo Trinchieri, MD2 Gian Luca Sfasciotti, MD, DDS, PhD1 Antonella Polimeni, MD, DDS1. Corresponding author: Prof. Antonella Polimeni Via A. Serranti, 49 - 00136 Rome, Italy Tel. +39 06 35453539 E-mail: antonella.polimeni@uniroma1.it. zi on al i. The arginine-deiminase enzymatic system on gingivitis: preliminary pediatric study. io. Summary The arginine-deiminase enzymatic system on gingivitis: preliminary pediatric study.. ©. C. IC. Ed. iz. Aim. The lactic bacteria are Gram-positive microorganisms with coccus or stick shape, which share a number of physiological and biochemical properties. Several experimental evidences suggest the possibility of using lactic acid bacterial as a preventive or therapeutic potential approaches, alternative or complementary to prevention or treatment protocols currently followed in several pathological conditions. Lactobacillus brevis is able, through the arginine-deiminase activity, to subtract the substrate (arginine) to nitric oxide synthase, and to inhibit in vitro generation of nitric oxide from rat’s peritoneal macrophages. These data led us to study the in vivo L. brevis anti-inflammatory effect choosing as experimental model the gingivitis. Materials and methods. In our study were examined 21 subjects, 16 males and 5 females, aged between 5 and 12 years, with marginal gingivitis problems who have been given chewing gum containing the principle to test in measure of three per day. Results. At the time T1, after treatment, 18 patients no longer showed inflammation; 2 of them had a slight inflammation and only 1 patient still showed a moderate inflammation. Conclusions. From our research, as confirmed by clinical and laboratory investigation, results an effective antiinflammatory action of arginine-deiminase system that some bacteria possessing. Key words: gingivitis, lactobacillus brevis, arginine-deiminase.. 8. Annali di Stomatologia 2010; I (1): 8-13.

(2) The arginine-deiminase enzymatic system on gingivitis: preliminary pediatric study. zi on al i. • To analyze the in vivo effects of the tablets containing L. brevis on clinical signs in patients with primitive and applicant marginal gingivitis. • To analyze the in vivo effects of the tablets containing L. brevis on the salivary IgA levels and on the nitric oxide synthase (NOS) activity (Fig. 1).. Materials and Methods. ni. In. • Personal data. • Accurate family history, physiological and pathological remote and next. • Possible presence of underlying conditions; excluding. subjects with systemic diseases or subjects under drug prolonged treatments. • Personal behavior including the possible presence of orthodontic appliances. They were selected subjects suffering from marginal gingivitis (Fig. 3) (T0) who have been given chewing gums containing the principle to test in three per day. The gums, taken at regular intervals, were chewed by patient for a period longer than 20 minutes. The checks were made at the time T1 (30 days) and T2 (60 days). During each control has been made a levy saliva. The saliva samples of patients and controls, collected in tubes before and after treatment and immediately frozen at -20°C until use, were analyzed for the activity of NOS (this was determined as levels of nitrites/nitrates in the hamlet of salivary fluid), and the levels of IgA (Giuca, 2007; Grbic, 1995; Haffajee, 1983; Heskens, 1996; Riccia, 2007).. rn a. Section 1. Figure 3 - Marginal gingivitis.. te. In our study were examined 21 subjects, 16 males (76%) and 5 females (24%), aged between 5 and 12 years, with marginal gingivitis problems, 16 volunteers as controls, 10 males (62.5%) and 6 females (37.5%), examined at the U.O.C. of Paediatric Dentistry of “ Sapienza”, University of Rome. The controls were used only for the evaluation of laboratory data, and not in clinical investigations. To patients were administered gums containing the active ingredient (Fig. 2) for a period of 60 days.The procedures were carried out in accordance with the Helsinki Declaration of 1975, and the 1983 revision of the same. For the study a medical bill in different sections was drawn up.. Ed. iz. io. Section 2. ©. C. IC. Figure 1 - Inhibition of NO-synthase by arginine-deiminase enzyme.. The clinical evaluation was performed referring on 6 dental elements (upper arch: earlier sector 1.1 or 1.2, medium-rear sector 1.6 and 2.6; lower arch: earlier sector 3.1 or 4.1, medium-rear sector 3.6 and 4.6), with symptoms (thermal sensitivity and bleeding) and objectivity (plaque, scale, gingival inflammation) recorded at the time T0, T1 and T2 above reported with a gradual scale (Tables 1 and 2).. Section 3 Registration at T1 and T2 of possible side effects resulting from the administration of the principle tested.. Section 4 Final evaluation including clinical and laboratory data. With regard to clinical aspects on the basis of results obtained has given judgment: • healing; • improvement; • failure; • relapse; • do not assessable.. Results. Figure 2 - Gums containing the active ingredients. Annali di Stomatologia 2010; I (1): 8-13. The clinical signs and symptoms (gingival inflammation, plaque, scale, sensitivity to temperature and bleeding after. 9.

(3) G. Ierardo et al.. mation and only 1 patient still showed a moderate inflammation. The tolerance to the treatment was considered very good in all patients. Overall dentist and patients opinion coincided and the results confirmed regression in 18 patients and improved in the remaining 3 of them. The effects of treatment on the levels of plaque and scale are shown in Table. zi on al i. pressure), given before and after treatment with the gums of L. brevis, are shown in Table 3 and Table 4. At the time T0, before treatment, gingival inflammation was evaluated moderate/diffused in 20 patients and slight in 1 patient (Table 3 and Figure 4). At the time T1, after treatment, 18 patients no longer showed inflammation; 2 of them had a slight inflam-. te. rn a. Table 1 - Symptoms with gradual scale shown in Section 2.. Ed. iz. io. ni. In. Table 2 - Signs objectives shown in Section 2.. ©. C. IC. Table 3 - Effect of gums with L. brevis on gingival inflammation, plaque and scale.. 10. Annali di Stomatologia 2010; I (1): 8-13.

(4) The arginine-deiminase enzymatic system on gingivitis: preliminary pediatric study. tients compared to the control samples, while treatment with tablets not producing a significant change.. zi on al i. Evaluation of nitrite/nitrate levels. rn a. Levels of nitrites/nitrates are often indicative of NOS activity. In our study we proposed to detect the activity of this enzyme. The results of determining the levels of nitrites and nitrates in the saliva of healthy subjects and subjects with gingivitis before (T0) and after (T1) treatment with the gums are reported in Tables 5 and 6 and in Figure 5. As it is possible to see levels of nitrites/nitrates in patients with gum disease were significantly higher (P<0.01) compared to those of controls. It seems clear that in all patients, treatment with the gums with L. brevis was associated with a significant reduction in the levels of nitrites and nitrates (P <0.01).. Conclusions. te. In. Figure 4 - Effect of gums with L. brevis on gingival inflammation, plaque and scale.. Lactobacillus brevis is able, through the arginine-deiminase activity, subtract the substrate (arginine) to nitric oxide synthase and to inhibit in vitro the generation of nitric oxide from rat’s peritoneal macrophages stimulated with LPS and IFNγ. These data led us to study in vivo the L. brevis anti-inflammatory effect choosing as experimental model the gingivitis. The choice was dictated by some observations and needs: L. brevis, like other lactic bacteria, is part of the normal flora of the oral cavity, is present in dental plaque and in literature is not reported as pathogen for periodontium; the method of collection of samples was not painful for patients and the inflammatory state and evolution of the disease were evaluated using an objective examination and with the use of a simple equipment. Overall, our results allow us to hypothesize that L. brevis has anti-inflammatory activity due to its ability to inhibit, in particular on macrophagic cells, the activity of iNOS, indirectly causing a reduction in levels of inflammatory cytokines. It was described the ability of some so-called protective microorganisms present in dental plaque, (for example Streptococcus sanguinis, Gram-positive, Veillonella parvula, Gramnegative, and Eubacteria), to suppress the growth of pathogenic bacteria. Therefore it would be advantageous to elim-. Evaluation of secretory IgA. io. ni. 3, which notes the improvement in the levels of plaque in almost all patients. In Table 4 (Figure 5) are shown the beneficial effects of treatment on sensitivity to temperature and on bleeding after pressure.. Ed. iz. They were analyzed levels of secretory IgA in saliva fluid fraction of control subjects and in patients with gingivitis before (T0) and after (T1) treatment with the gums. In Tables 5 and 6 and in Figure 6 are showed the data about antibody. A significant reduction in IgA levels was founded in saliva of the pa-. ©. C. IC. Table 4 - Effect of gums with L. brevis on sensitivity to temperature and bleeding.. Annali di Stomatologia 2010; I (1): 8-13. 11.

(5) G. Ierardo et al.. zi on al i. Table 6 - Levels of nitrites/ nitrates and IgA s in patients with gingivitis before (T0) and after (T1) treatment with L. brevis gums.. Data displayed as ± STD. *P < 0.05 mean.. rn a. In. Table 5 - Levels of nitrites/ nitrates, PGE2, cytokine and IgAs in controls and patients with periodontitis before (T0) treatment with L. brevis gums.. te. Figure 5 - Effect of gums with L. brevis on sensitivity to temperature and bleeding.. abling an efficient competition with the same bacteria pathogens for periodontium for membership of adhesion sites on dental and epithelial surfaces. From our research, as confirmed by clinical and laboratory investigation, results an effective anti-inflammatory action of arginina-deiminasi system, that some bacteria possessing. It is conceivable that the anti-inflammatory action of argininadeiminasi system can also act in other inflammatory conditions, not necessarily where there is a bacterial component at the base, acting mainly through iNOS inhibition. This is the case for example of recurring aphthosus stomatitis, multifactorial etiology disease, for which there isn’t still a valid treatment without side effects. Precisely for this inflammatory condition, we are implementing a therapeutic and research protocol, which aims to assess the efficacy of “arginine-deiminase” enzymatic system.. ni. References. io. 1.. ©. C. IC. Ed. iz. Data displayed as ± STD. *P < 0.05 mean.. Figure 6 - Salivary IgA level of controls and patients with gingivitis marginalis, before (T0) and after (T1) treatment with L. brevis gums.. inate harmful bacteria, encouraging the growth of beneficial microorganisms. We can not exclude that the administration of L. brevis, normally present in the oral flora and plaque, en-. 12. Bucci P, Bucci M, Scutellà M, Giannone N, Santarelli A. Microbiological agents associated with IL-1 polymorphisms in periodontal disease. A pilot study Annali di Stomatologia 2006;LV(1):23-29. 2. Chambers DA, Imrey PB, Cohen RL. A cross-selectional analysis of aspartate aminotransferase in gingival crevicular fluid. J Periodontal Res. 1991; 26: 75-84. 3. Chambers DA, Imrey PB, Cohen RL. A longitudinal study of aspartate aminotransferase in human gingival crevicular fluid. J Periodontal Res. 1991; 26:65-74. 4. De Luca M, Dolci G, Passariello C. Gli anaerobi e la malattia parodontale. Bologna: Martina Ed; 1999. 5. Giuca MR, Saracino S, Giannotti E, Ceccarini A. Oral clearance of NaF from chewing gum and tablets in children and adults. Eur J Paediatr Dent. 2007; Mar;8(1):19-24. 6. Grbic JT, Singer RE, Jans HH, Celenti RS, Lamster BI. Immunoglobulin Isotypes in gingival crevicular fluid: possible protective role of IgA. J Periodontol. 1995; 66:55-61. 7. Haffajee AD, Socransky SS, Goodson JM. Clinical parameters as predictors of destructive periodontal disease activity. J Clin Periodontol. 1983; 10: 257-265. 8. Henskens YMC, Van den Keijbus PAM, Veerman ECI, Van der Weijden GA, Timmermann MF, Snoek CM, Van der Velden U. Nieuw Amerongen A.V.: Protein composition of whole and parotid saliva in healthy and periodontitis subjects. J Periodontal Res. 1996; 31:57-65. 9. Lamster JB, Harper DS, Fiorello LA. Lisosomial and cytoplasmic enzime activity, crevicular fluid volume, and clinical parameters characterizing gingival sites with shallow to intermediate probing depths. J Periodontol. 1987; 58: 614-62. 10. Lindhe J, Karring T, Lang NP. Parodontologia e Implantologia Dentale. Milano: Ermes Ed; 1998. 11. Malin M, Souomalainen H, Saxelin M, Isolauri E. Promotion of IgA immune. Response in patients with Crohn’s disease by oral bacteriotherapy with Lactobacillus GG. Ann Nutr Metab. 1996; 40:137-45. 12. Mizuho F, Mori H, Deguchi S. Aspartate aminotransferase levels in human periodontum derived cells. J Periodontol. Annali di Stomatologia 2010; I (1): 8-13.

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