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

2. The arginine-deiminase enzymatic system on gingivitis: preliminary pediatric study
• To analyze the in vivo effects of the tablets containing L. bre-
vis on clinical signs in patients with primitive and applicant
marginal 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).
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Materials and Methods
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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
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controls were used only for the evaluation of laboratory data, Figure 3 - Marginal gingivitis.
and not in clinical investigations. To patients were administered
gums containing the active ingredient (Fig. 2) for a period of subjects with systemic diseases or subjects under drug pro-
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60 days.The procedures were carried out in accordance with longed treatments.
the Helsinki Declaration of 1975, and the 1983 revision of the • Personal behavior including the possible presence of or-
same. For the study a medical bill in different sections was thodontic appliances.
drawn up. They were selected subjects suffering from marginal gingivi-
tis (Fig. 3) (T0) who have been given chewing gums containing
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the principle to test in three per day. The gums, taken at reg-
Section 1 ular intervals, were chewed by patient for a period longer than
20 minutes. The checks were made at the time T1 (30 days)
• Personal data. and T2 (60 days). During each control has been made a levy
• Accurate family history, physiological and pathological re-
mote and next.
• Possible presence of underlying conditions; excluding
nt saliva. The saliva samples of patients and controls, collect-
ed 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
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salivary fluid), and the levels of IgA (Giuca, 2007; Grbic, 1995;
Haffajee, 1983; Heskens, 1996; Riccia, 2007).
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Section 2
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, medi-
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um-rear sector 3.6 and 4.6), with symptoms (thermal sensi-
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tivity and bleeding) and objectivity (plaque, scale, gingival in-
flammation) recorded at the time T0, T1 and T2 above reported
with a gradual scale (Tables 1 and 2).
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Section 3
Registration at T1 and T2 of possible side effects resulting from
Figure 1 - Inhibition of NO-synthase by arginine-deiminase en-
the administration of the principle tested.
zyme.
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Section 4
Final evaluation including clinical and laboratory data. With re-
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gard to clinical aspects on the basis of results obtained has
given judgment:
• healing;
• improvement;
• failure;
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• relapse;
• do not assessable.
Results
The clinical signs and symptoms (gingival inflammation,
Figure 2 - Gums containing the active ingredients. plaque, scale, sensitivity to temperature and bleeding after
Annali di Stomatologia 2010; LIX (1): 8-13 9

3. G. Ierardo et al.
pressure), given before and after treatment with the gums of mation and only 1 patient still showed a moderate inflam-
L. brevis, are shown in Table 3 and Table 4. At the time T0, mation. The tolerance to the treatment was considered very
before treatment, gingival inflammation was evaluated mod- good in all patients. Overall dentist and patients opinion co-
erate/diffused in 20 patients and slight in 1 patient (Table 3 incided and the results confirmed regression in 18 patients
and Figure 4). At the time T1, after treatment, 18 patients no and improved in the remaining 3 of them. The effects of treat-
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longer showed inflammation; 2 of them had a slight inflam- ment on the levels of plaque and scale are shown in Table
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Table 1 - Symptoms with gradual scale shown in Section 2.
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Table 2 - Signs objectives shown in Section 2.
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Table 3 - Effect of gums with L. brevis on gingival inflammation, plaque and scale.
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10 Annali di Stomatologia 2010; LIX (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.
Evaluation of nitrite/nitrate levels
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Levels of nitrites/nitrates are often indicative of NOS activity.
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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
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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 lev-
els of nitrites/nitrates in patients with gum disease were sig-
nificantly higher (P<0.01) compared to those of controls. It
seems clear that in all patients, treatment with the gums with
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L. brevis was associated with a significant reduction in the lev-
els of nitrites and nitrates (P <0.01).
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Conclusions
Lactobacillus brevis is able, through the arginine-deiminase
activity, subtract the substrate (arginine) to nitric oxide syn-
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thase and to inhibit in vitro the generation of nitric oxide from
rat’s peritoneal macrophages stimulated with LPS and IFN-
γ. These data led us to study in vivo the L. brevis anti-in-
Figure 4 - Effect of gums with L. brevis on gingival inflammation, flammatory effect choosing as experimental model the gin-
plaque and scale.
3, which notes the improvement in the levels of plaque in al-
nt givitis. The choice was dictated by some observations and
needs: L. brevis, like other lactic bacteria, is part of the nor-
mal flora of the oral cavity, is present in dental plaque and in
literature is not reported as pathogen for periodontium; the
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most all patients. In Table 4 (Figure 5) are shown the bene- method of collection of samples was not painful for patients
and the inflammatory state and evolution of the disease were
ficial effects of treatment on sensitivity to temperature and on
evaluated using an objective examination and with the use of
bleeding after pressure.
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a 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 of
Evaluation of secretory IgA iNOS, indirectly causing a reduction in levels of inflammato-
ry cytokines.
They were analyzed levels of secretory IgA in saliva fluid frac-
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It was described the ability of some so-called protective mi-
tion of control subjects and in patients with gingivitis before
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croorganisms present in dental plaque, (for example Strep-
(T0) and after (T1) treatment with the gums. In Tables 5 and tococcus sanguinis, Gram-positive, Veillonella parvula, Gram-
6 and in Figure 6 are showed the data about antibody. A sig- negative, and Eubacteria), to suppress the growth of patho-
nificant reduction in IgA levels was founded in saliva of the pa- genic bacteria. Therefore it would be advantageous to elim-
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Table 4 - Effect of gums with L. brevis on sensitivity to temperature and bleeding.
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Annali di Stomatologia 2010; LIX (1): 8-13 11

5. G. Ierardo et al.
Table 6 - Levels of nitrites/ nitrates and IgA s in patients with
gingivitis before (T0) and after (T1) treatment with L. brevis
gums.
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Data displayed as ± STD. *P < 0.05 mean.
abling an efficient competition with the same bacteria
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pathogens for periodontium for membership of adhesion sites
on dental and epithelial surfaces.
From our research, as confirmed by clinical and laboratory in-
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vestigation, results an effective anti-inflammatory action of
arginina-deiminasi system, that some bacteria possessing. It
is conceivable that the anti-inflammatory action of arginina-
deiminasi system can also act in other inflammatory conditions,
Figure 5 - Effect of gums with L. brevis on sensitivity to temper- not necessarily where there is a bacterial component at the
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ature and bleeding. 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 with-
Table 5 - Levels of nitrites/ nitrates, PGE2, cytokine and IgAs out side effects.
in controls and patients with periodontitis before (T0) treat-
ment with L. brevis gums.
nt Precisely for this inflammatory condition, we are implement-
ing a therapeutic and research protocol, which aims to assess
the efficacy of “arginine-deiminase” enzymatic system.
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