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

Gaetano Ierardo, DDS, PhD1

Maurizio Bossù, DDS, PhD1

Desirée Tarantino, DDS1

Vincenzo Trinchieri, MD2

Gian Luca Sfasciotti, MD, DDS, PhD1

Antonella Polimeni, MD, DDS1

“Sapienza” University of Rome, Rome, Italy1 Department of Oral Sciences, Paediatric Dentistry Unit,(A. Polimeni)2 Department of Infectious and Tropical Diseases (V. Vullo)

Corresponding author: Prof. Antonella PolimeniVia A. Serranti, 49 - 00136 Rome, ItalyTel. +39 06 35453539E-mail: [email protected]

SummaryThe arginine-deiminase enzymatic system on gingivitis:preliminary pediatric study.

Aim. The lactic bacteria are Gram-positive microorgan-isms with coccus or stick shape, which share a numberof physiological and biochemical properties. Several ex-perimental evidences suggest the possibility of usinglactic acid bacterial as a preventive or therapeutic po-tential approaches, alternative or complementary to pre-vention or treatment protocols currently followed inseveral pathological conditions.Lactobacillus brevis is able, through the arginine-deim-inase activity, to subtract the substrate (arginine) to ni-tric oxide synthase, and to inhibit in vitro generation ofnitric oxide from rat’s peritoneal macrophages. Thesedata led us to study the in vivo L. brevis anti-inflamma-tory effect choosing as experimental model the gingivi-tis. Materials and methods. In our study were examined 21subjects, 16 males and 5 females, aged between 5 and12 years, with marginal gingivitis problems who havebeen given chewing gum containing the principle to testin measure of three per day. Results. At the time T1, after treatment, 18 patients nolonger showed inflammation; 2 of them had a slight in-flammation and only 1 patient still showed a moderateinflammation.Conclusions. From our research, as confirmed by clini-cal and laboratory investigation, results an effective anti-inflammatory action of arginine-deiminase system thatsome bacteria possessing.Key words: gingivitis, lactobacillus brevis, arginine-deimi-nase.

Introduction

The lactic bacteria are Gram-positive microorganisms with coc-cus or stick shape, which share a number of physiological andbiochemical properties. They include some species of lacto-bacilli, lattococchi, streptococci, pediococchi. Several exper-imental evidences suggest the possibility of using lactic acidbacteria as a potential preventive or therapeutic approaches,alternative or complementary protocols prevention or treatmentcurrently followed in several pathological conditions (Offen-bacher, 1993; Mombelli, 1998) Specific components of Lac-tobacilli seem to have an intense effect on the modulation ofimmune response, on the activation of the reticuloendothe-lial system and on the regulating production of cytokines andinflammatory response (Malin, 1996; Naidu, 1999). In this wayit is very important to the study conducted by Ulisse (Ulisse,2001) in which was found an inhibitory action of Lactobacil-lus brevis against iNOS, in rat’s peritoneal macrophages stim-ulated in vitro with IFN-γ and lipopolysaccharide (LPS). In thelight of these results, in this work, we have proposed to as-sess the anti inflammatory effects of Lactobacillus brevis incase of marginal gingivitis. The initial pathogenic event de-termines in guest’s organism tissues the production and syn-thesis of cytokines (Bucci, 2006). The excessive productionof cytokines driving in a non-specific way the production of ni-tric oxide (NO).NO2, gaseous free radical, is fleeting and in the presence ofoxygen is rapidly metabolized by nitrate to nitrite (NO). Theincrease of NO can not be seen as specific for a clinical con-dition, but rather as a reflection of an immuno-inflammatoryactivated state, in which cytokines, together with other me-diators, led to a stimulation of NO-synthase in tissues and flu-ids (Ramberg, 1994; Persson, 1990).It is clear that excessive levels of NO are harmful and dan-gerous for the tissues and promote the progression of the in-flammatory phenomena (De Luca, 1999). The plaque bacte-ria that have arginina-deiminase enzyme are: Streptococcussanguinis, Streptococcus milleri, Streptococcus rattus, Lac-tobacillus fermentum, Streptococcus faecalis, Streptococcusfaecium, Eubacterium nodatum species. Some strains of Lac-tobacillus brevis species (CD2) are surprisingly rich in “argin-ina-deiminasi” enzymatic system. It is believed that the enzymemay play an important role in the ecology of the mouth prob-ably protecting oral bacteria that are less tolerant against acidenvironment, by the pH decrease (4.0) caused by aci-dophilous bacteria glycolysis (Lindhe, 1998; Page, 1996; Poli-meni, 1995). This defense is expressed through an increasein pH associated with the production of ammonia. The actionpad done by ammonia can be positive not only for the sameplaque bacteria, but also for tooth and tissues of the guest.This enzyme uses the same iNOS substrate, inhibiting its ac-tion, and pursuing anti-inflammatory effects (Chambers,1991; Lamster, 1987; Wong, 1999; Mizuho, 1996).The goal of the study has been to identify the possible med-ical applications, both preventively and therapeutically, of thelactic acid bacteria strains selected on the basis of specificchemical, physical and/or biological characteristics.In particular, objectives of this work were:

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

8 Annali di Stomatologia 2010; LIX (1): 8-13

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Annali di Stomatologia 2010; LIX (1): 8-13 9

• To analyze the in vivo effects of the tablets containing L. bre-vis on clinical signs in patients with primitive and applicantmarginal gingivitis.

• To analyze the in vivo effects of the tablets containing L. bre-vis on the salivary IgA levels and on the nitric oxide syn-thase (NOS) activity (Fig. 1).

Materials and Methods

In our study were examined 21 subjects, 16 males (76%) and5 females (24%), aged between 5 and 12 years, with marginalgingivitis problems, 16 volunteers as controls, 10 males(62.5%) and 6 females (37.5%), examined at the U.O.C. ofPaediatric Dentistry of “ Sapienza”, University of Rome. Thecontrols were used only for the evaluation of laboratory data,and not in clinical investigations. To patients were administeredgums containing the active ingredient (Fig. 2) for a period of60 days.The procedures were carried out in accordance withthe Helsinki Declaration of 1975, and the 1983 revision of thesame. For the study a medical bill in different sections wasdrawn up.

Section 1

• Personal data.• Accurate family history, physiological and pathological re-

mote and next.• Possible presence of underlying conditions; excluding

subjects with systemic diseases or subjects under drug pro-longed treatments.

• Personal behavior including the possible presence of or-thodontic appliances.

They were selected subjects suffering from marginal gingivi-tis (Fig. 3) (T0) who have been given chewing gums containingthe principle to test in three per day. The gums, taken at reg-ular intervals, were chewed by patient for a period longer than20 minutes. The checks were made at the time T1 (30 days)and T2 (60 days). During each control has been made a levysaliva. The saliva samples of patients and controls, collect-ed in tubes before and after treatment and immediately frozenat -20°C until use, were analyzed for the activity of NOS (thiswas determined as levels of nitrites/nitrates in the hamlet ofsalivary fluid), and the levels of IgA (Giuca, 2007; Grbic, 1995;Haffajee, 1983; Heskens, 1996; Riccia, 2007).

Section 2

The clinical evaluation was performed referring on 6 dentalelements (upper arch: earlier sector 1.1 or 1.2, medium-rearsector 1.6 and 2.6; lower arch: earlier sector 3.1 or 4.1, medi-um-rear sector 3.6 and 4.6), with symptoms (thermal sensi-tivity and bleeding) and objectivity (plaque, scale, gingival in-flammation) recorded at the time T0, T1 and T2 above reportedwith a gradual scale (Tables 1 and 2).

Section 3

Registration at T1 and T2 of possible side effects resulting fromthe administration of the principle tested.

Section 4

Final evaluation including clinical and laboratory data. With re-gard to clinical aspects on the basis of results obtained hasgiven judgment:• healing;• improvement;• failure;• relapse;• do not assessable.

Results

The clinical signs and symptoms (gingival inflammation,plaque, scale, sensitivity to temperature and bleeding after

Figure 3 - Marginal gingivitis.

Figure 2 - Gums containing the active ingredients.

Figure 1 - Inhibition of NO-synthase by arginine-deiminase en-zyme.


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pressure), given before and after treatment with the gums ofL. brevis, are shown in Table 3 and Table 4. At the time T0,before treatment, gingival inflammation was evaluated mod-erate/diffused in 20 patients and slight in 1 patient (Table 3and Figure 4). At the time T1, after treatment, 18 patients nolonger showed inflammation; 2 of them had a slight inflam-

mation and only 1 patient still showed a moderate inflam-mation. The tolerance to the treatment was considered verygood in all patients. Overall dentist and patients opinion co-incided and the results confirmed regression in 18 patientsand improved in the remaining 3 of them. The effects of treat-ment on the levels of plaque and scale are shown in Table

Table 1 - Symptoms with gradual scale shown in Section 2.

Table 2 - Signs objectives shown in Section 2.

Table 3 - Effect of gums with L. brevis on gingival inflammation, plaque and scale.

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3, which notes the improvement in the levels of plaque in al-most all patients. In Table 4 (Figure 5) are shown the bene-ficial effects of treatment on sensitivity to temperature and onbleeding after pressure.

Evaluation of secretory IgA

They were analyzed levels of secretory IgA in saliva fluid frac-tion of control subjects and in patients with gingivitis before(T0) and after (T1) treatment with the gums. In Tables 5 and6 and in Figure 6 are showed the data about antibody. A sig-nificant reduction in IgA levels was founded in saliva of the pa-

tients compared to the control samples, while treatment withtablets not producing a significant change.

Evaluation of nitrite/nitrate levels

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 nitratesin the saliva of healthy subjects and subjects with gingivitisbefore (T0) and after (T1) treatment with the gums are reportedin Tables 5 and 6 and in Figure 5. As it is possible to see lev-els of nitrites/nitrates in patients with gum disease were sig-nificantly higher (P<0.01) compared to those of controls. Itseems clear that in all patients, treatment with the gums withL. brevis was associated with a significant reduction in the lev-els of nitrites and nitrates (P <0.01).

Conclusions

Lactobacillus brevis is able, through the arginine-deiminaseactivity, subtract the substrate (arginine) to nitric oxide syn-thase and to inhibit in vitro the generation of nitric oxide fromrat’s peritoneal macrophages stimulated with LPS and IFN-γ. These data led us to study in vivo the L. brevis anti-in-flammatory effect choosing as experimental model the gin-givitis. The choice was dictated by some observations andneeds: L. brevis, like other lactic bacteria, is part of the nor-mal flora of the oral cavity, is present in dental plaque and inliterature is not reported as pathogen for periodontium; themethod of collection of samples was not painful for patientsand the inflammatory state and evolution of the disease wereevaluated using an objective examination and with the use ofa simple equipment. Overall, our results allow us to hypoth-esize that L. brevis has anti-inflammatory activity due to its abil-ity to inhibit, in particular on macrophagic cells, the activity ofiNOS, indirectly causing a reduction in levels of inflammato-ry cytokines.It was described the ability of some so-called protective mi-croorganisms present in dental plaque, (for example Strep-tococcus sanguinis, Gram-positive, Veillonella parvula, Gram-negative, and Eubacteria), to suppress the growth of patho-genic bacteria. Therefore it would be advantageous to elim-

Figure 4 - Effect of gums with L. brevis on gingival inflammation,plaque and scale.

Table 4 - Effect of gums with L. brevis on sensitivity to temperature and bleeding.

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inate harmful bacteria, encouraging the growth of beneficialmicroorganisms. We can not exclude that the administrationof L. brevis, normally present in the oral flora and plaque, en-

abling an efficient competition with the same bacteriapathogens for periodontium for membership of adhesion siteson dental and epithelial surfaces.From our research, as confirmed by clinical and laboratory in-vestigation, results an effective anti-inflammatory action ofarginina-deiminasi system, that some bacteria possessing. Itis conceivable that the anti-inflammatory action of arginina-deiminasi system can also act in other inflammatory conditions,not necessarily where there is a bacterial component at thebase, acting mainly through iNOS inhibition. This is the casefor example of recurring aphthosus stomatitis, multifactorialetiology disease, for which there isn’t still a valid treatment with-out side effects. Precisely for this inflammatory condition, we are implement-ing a therapeutic and research protocol, which aims to assessthe efficacy of “arginine-deiminase” enzymatic system.

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Table 6 - Levels of nitrites/ nitrates and IgA s in patients withgingivitis before (T0) and after (T1) treatment with L. brevisgums.

Data displayed as ± STD. *P < 0.05 mean.

Figure 5 - Effect of gums with L. brevis on sensitivity to temper-ature and bleeding.

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Data displayed as ± STD. *P < 0.05 mean.

Figure 6 - Salivary IgA level of controls and patients with gingivi-tis marginalis, before (T0) and after (T1) treatment with L. brevisgums.© CIC

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Health & Medicine

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