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La inoculacion con Mycobacterium phlei inhibe la inflamacion alergica en un modelo de conejos sensibilizados con ovoalbumina (OVA).


In the last decades, the prevalence of atopic disease and other TH2 autoimmune disorders have increased in Western countries (1,8,35). One theory explains that fact by the hygiene hypothesis, which suggests an altered exposure to microbes in the environment, due to improved sanitation and personal hygiene, smaller family sizes, shorter duration of breastfeeding, immunizations and lack of serious childhood infections results in alteration of immunoregulation (24,32,34). This hypothesis is based on an inbalance in T-helper (Th) type response and Th cell-regulatory mechanisms due to the lack of microbial stimulation (14,25,31).

In fact, some authors describes an increased rate of allergy due to reduced exposure to environmental mycobacteria or mycobacterial products (14,34). In mice BCG vaccination leads to Th1 profile in airway allergic responses (reduce levels of immunoglobulin E, eosinophils, interleukin-4, and interleukin-5) to ovalbumin sensitization (10). Inoculation of M. vaccae or BCG reduced asthmatic responses in mice (15,38). A similar pattern is observed in rats with BCG (a suppression of atopic symptoms with a reduced TH2 responses, IgE and Il-4) (19,20,21).

In a mouse model intranasal application of BCG reduced eosinophil numbers and TH2 response to ovalbumin (10,24). In some animal models it is already demonstrated that mycobacteria can restore immune homeostasis (4). Mycobacteria are Gram-positive aerobic bacteria belonging to the family Mycobacteriaceae and are one of several mycolic-acid-containing genera within the order Actinomycetales (17,29). M. phlei, a commensal Mycobacterium non pathogenic for rabbit is widely distributes in the nature and has been used as adjuvant for different kinds of vaccines (4).

We have previously developed a rabbit model of allergic inflammation in rectal mucosa after ovalbumin challenge in sensitized animals33. Rabbit (Oryctolagus cuniculus) is one of the animal species frequently employed as an experimental model in human and veterinary research (11,23).

The aim of the present study is to determine the effect of M. phlei inoculation on allergic response from sensitized rabbits at early stages after birth.

Materials and methods

Thirty two adult New Zealand rabbits were divided in 4 groups:

1. Group 1: (n=8) Normal control.

2. Group 2: (n=8) Ovalbumin (OVA) sensitized subcutaneously (SC).

3. Group 3: (n=8) OVA sensitized SC and OVA rectal challenged.

4. Group 4: (n=8) OVA sensitized SC and OVA rectal challenged previously inoculated with M. phlei at days 1, 30 and 60 after birth. M. plhei inoculation was developed by subcutaneous administration of 3x107 bacilli per rabbit (final volume 0,1ml).

As we described in previous works, rabbits from group 2, 3 and 4 were twice subcutaneously sensitized with 70 [micro]g OVA (Grade V Sigma) in 30 mg ALUM/ml (aluminium hydroxide)2, 3.

An interval of 15 days among sensitizations was developed. After 15 days post sensitization, rabbits were 24 hours fasten and G3 and G4 animals were rectal challenged with 50 mg OVA in 5 ml of Phosphate

Buffered Saline (PBS).

Animals were sacrificed with sulfuric ether 4 hours after challenged, according to considerations of Ethical Cometee of Rosario School of Medicine and samples from rectum were obtained from all groups. Material for immunohistochemistry were snap frozen in liquid nitrogen with OCT protector and stored at -20 [grados]C and cut at 8 micrometers.

Samples for histology were paraffin embedded and cut at 6 micrometers.

The following monoclonal antibodies were used:

1. Mouse anti-rabbit CD4 (KEN-4, BALB/c IgG 2a, AbD SEROTEC, UK) (22).

2. Mouse anti-rabbit CD8 (12.C7, BALB/c IgG1, AbD SEROTEC, UK) (7).

3. Mouse anti-rabbit CD25 ((Kei-a1BALB/c IgG2b, AbD SEROTEC, UK) (22).

Botin-streptavidin system (Sero tec Ltd. Oxford, UK) was used for detection of marked cells and AEC as chromogen. Frozen samples were fixed in cold acetone for 10 minutes and then incubate for 60 minutes at 37 [grados]C with monoclonal antibodies. Cromotope II technique was employed to detection of eosinophils and Alcian blue pH < 1 was used for mast-cells in rectum.

Positive cells were analyzed in 200 high power fields in each group and expressed as cell mean per microscopical field by morphometric analysis9. Results were statistical analyzed.

Specific anti-OVA IgE titres were evaluated by passive cutaneous anaphylaxis PCA (5).

The results were expressed as mean. The comparison between variables in different experimental groups was made by analysis of variance by ranks of Kruskal-Wallis or Mann-Whitney U test, using the statistical program Medcalc 3.6.

Samples were considered different when they had a significance level <0.05.


Specific anti-OVA-IgE levels were evaluated by positive passive cutaneous anaphylaxis test (PCA) possitive at 160 fold dilutions in sensitized groups (G2, G3, G4). Histology of samples showed patchy mucosal edema, lymphangiectasia and eosinophil infiltration in the experimental OVA sensitized and challenged groups (G3 and G4). Eosinophils were spread on the mucosa underlying the epithelium. There were no visualized histopathological modifications in the control (Gl) and the sensitized and unchallenged group (G2). Different cell population quantification were summarized in table 1.

CD4, CD8 and CD25 positive cells from different groups were visualized in figures 1, 2, 3 and 4.






Ovalbumin is a soluble antigen which, when subcutaneously administered, induces sensitization that elicits specific IgE antibodies (37). Challenge with OVA in sensitized individuals produces an anaphylactic allergic response in the digestive tract. In previous works we found modifications in number of enteroendocrine cells, mast cells and eosinophils in rectal mucosa from OVA-sensitized and challenged rabbit (3,11,37). These changes associated to high levels of specific anti-OVA IgE, indicated an immediate hypersensivity reaction.

In the present work we observed eosinophil infiltration, vascular congestion and lymphangiectasia in group 2 and 3 (sensitized and challenged). Important strengths of our study are the objective histopathological findings of allergic inflammation in rectal mucosa in sensitized animals.

Such signs become apparent 4 hours after OVA challenge. In reference to M. Phlei inoculated rabbits, our study demonstrated an inhibition of allergic inflammation in rectal mucosa vs group 2 and 3.

As seen in results referred to reduced eosinophil infiltration and mast cell degranulation in rabbits inoculated with M. phlei we conclude that early contact with mycobacteria inhibits in some way allergic infiltration and inflammation. These results are similar to that found by different authors at airways in mice (15,38,39). We demonstrated a statistic significant increase in number of CD4+ cellular populations in animals from G2 and G4 as compared to control. Sensitization and oral challenge induce certain immune cells populations recruitment and migrationphenomenawithadecreaseinnaivelymphocytes.

Conversely the immune activation of T cells is evidenced by an enhance of CD25 (interleukin-2 receptor) membrane receptor in sensitized and challenged rabbits (G3), these increase is strongly inhibited in M. Phlei inoculated animals (G4). These data is similar to that found by Saavedra et al. (30), showing evidence that mycobacterial cell-surface glycolipid are able to reduce antigen-induced proliferation of human CD4+ and CD8+ T-cell subsets associated with a decrease of cells expressing the T-cell surface activation markers CD25 and CD69.

Different mechanisms could explain these findings. Exposition to mycobacteria at early stages of life may downregulate allergic response through the induction of T regulatory cells and production of anti-inflammatory cytokines (12,13). Some authors have demonstrated that simultaneous mycobacteria inoculation and allergen sensitization may stimulate a Th1 profile able to suppress Th2 activity significantly18.

In our model stimulation of T cells with OVA by subcutaneous sensitization only induced Th2-dominant responses, whereas concomitant stimulation with both

OVA and M. plhei could caused induction of antigen- specific Th1 responses, with secretion of IFN-g, mediated by IL-12 as described by C. Obihara (27,28).

Based on the evidence presented in this work, we conclude that M. phlei (or M. phlei products) might be able to protect against the development of allergic inflammation and atopic disease. Although the exact mechanisms involved in the protective effect are not yet known, immune mechanisms involved in the defense against Mycobacteria and immune modulation could be reasons for observed effects on this animal model.

(Recibido: 24 de junio, 2010; aceptado: 17 de noviembre, 2010)


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* Corresponding for author: Dr. Vinuesa, Miguel. Superi 247, Rosario (2000). Fax: 54-341-425-5236, Santa Fe, Argentina. E-mail:

Miguel Angel Vinuesa [1,*], MD, PhD; Norberto David Bassan [1], MD; Ana Ines Cases [1], MD; Gustavo Krumrick [1], MD; Soledad Chaparro [1], MD.

[1] Catedra de Histologia y Embriologia, Facultad de Ciencias Medicas, Universidad Nacional de Rosario.Santa Fe 3100, Rosario (2000), Argentina.
Table 1. Cell populations in rectal mucosa from normal
rabbits, ova sensitized rabbits and sensitized and locally
challenged rabbits. Positive cells per high power field
arithmetic mean and SE.

                G1: Control        G2: sensirized
                  N = 200             n = 200


CD 4         8,35 [+ or -] 0,7   13,4 [+ or -] 0,9
CD 8         6,28 [+ or -] 0,5   8,25 [+ or -] 0,8
CD 25        13,2 [+ or -] 1,2   15,1 [+ or -] 1,9
Mast cells    8,7 [+ or -] 1,3   17,6 [+ or -] 2,5
Eosinophs     1,2 [+ or -] 0,6    3,1 [+ or -] 0,2

              G3: sensitized      G4: sensitized
              and challenged      and challenged,
Markers           n = 200         treated with M. phlei
                                      n = 200

CD 4          8,26 [+ or -] 0,6   17,26 [+ or -] 1,2 *
CD 8         10,77 [+ or -] 0,5   11,29 [+ or -] 1,1
CD 25           20 [+ or -] 1,4   14,41 [+ or -] 1,9 *
Mast cells     6,0 [+ or -] 1,8     7,2 [+ or -] 1,7 **
Eosinophs     13,1 [+ or -] 0,8    1,33 [+ or -] 0,4 **

* Different from G2 and G3 p<0,05. ** Different from G2 (p<0,001). *
Different from G3 (p<0,05). ** Different from G3 (p<0,05)
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Title Annotation:Articulos orginales de investigacion
Author:Vinuesa, Miguel Angel; Bassan, Norberto David; Cases, Ana Ines; Krumrick, Gustavo; Chaparro, Soledad
Publication:Revista CES Medicina Veterinaria y Zootecnia
Date:Jul 1, 2010
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