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EXPERIMENTAL CHRONIC TUBERCULO-TYPHOID EXPOSURE IN A LAPIAN IMMUNE EVALUATION MODEL II: CUMULATED EFFECT


الناشر \ المحرر \ الكاتب (Author / Editor / Publisher)

 
زينب خضر احمد المهدي الامين

Citation Information


زينب,خضر,احمد,المهدي,الامين , EXPERIMENTAL CHRONIC TUBERCULO-TYPHOID EXPOSURE IN A LAPIAN IMMUNE EVALUATION MODEL II: CUMULATED EFFECT , Time 08/12/2015 11:17:57 : كلية طب الاسنان

وصف الابستركت (Abstract)


To evaluate the possible influences of chronic cumulated exposure to lower concentrations of tuberculin

الوصف الكامل (Full Abstract)

ABSTRACT
Aim of the Study: To evaluate the possible influences of chronic cumulated exposure to lower concentrations of tuberculin on the immune response to typhoid antigen in a lapin model. Methods: Residual tuberculin concentration of 0.00005 ,0.0005, 0.005 and 0.05 IU were nasally applied through aerosolizer in a week wise manner to ten newzeland white rabbits, five of which were left four days then immunized with S. typhi “O” antigen (group I). The other five however were left as such for one more week (Group II). Ten other neuzland white rabbits were equally subdivided in to S. typhi immunized rabbits (Group III) and saline injected rabbits as control (Group IV). A set of immune function test were performed to the four groups of theserabbits. Result: The cumulated tuberculin exposure (Group II) lead to increase of NBT neutrophil phagocytosis ; significant LIF percentage; E.rossete count; IL-1?, IL-12 and IL- 10 but not IL-4 and skin DTH. While combined Tuberculo-Typhoid exposure (Group I) lead to increase in NBT, E.rosette and IgM anti S. typhi “O” but not DTH. Both of the exposure modalities shifts cytokine balance towards proinflammatory cytokines. Conclusion: The major immune feature of these exposure modalities was: 1- Immunomodulation, 2-T-Cell competition, 3-Cytokine unbalance and 4- inhibition of IgM class switching to IgG.
INTRODUCTION
Chronic intracellular bacterial infections may release residiual bacterial proteins within the vertebrate host like man. Such release can be immunotoxic to the host [1]-[5]. In the present work residual tuberculin concentration increasing up to 0.05 IU were nasally applied through aerosolizer followed by four days relief then immunized with two oral then after two intravenous doses of S. typhi in a lapian model.
MATERIALS AND METHODS
1-Tuberculin
Tubersol is a sterile isotonic solution of Tuberculin (Purified protein derivative of M. tuberculosis (5TU per 0.1 ml) in phosphate buffered saline containing Tween 80 (0.0005%) as a stabilizer. Phenol 0.28% is added as a preservative [6], [7]. Four sequenced concentrations (0.05TU, 0.005TU, 0.0005 TU& 0.00005TU) were made in phosphate buffer saline.
2-S. typhi ”O”
antigen (bacterin) as heat killed antigen was prepared as [8].
3-S.typhi Sensitine
Sensitine is cell free culture filtrate. It was prepared as 24hr culture in 0.5 % glucose peptone water. Growth centrifuged at 2500 rpm for 5 min. Supernatant was filtrated through 0.22 mm. membrane filter. [9].
4-Rabbits
Twenty male newzland rabbits were of 1-1.5 kg body weight kept a libitum and housed in individual wire-rod floored and stain-less steal cages, each measuring 48 x 16 x 46 cm with collection Pan beneath each gage. They were grouped into four groups each of five as in the follow diagram Fig-1.
5-Tuberculin Inhalation Technique
Inhalation was performed using compressor nebulizer (Mabie Co .England). It passes aerosol containing tuberculin of 0.5 to one mm size for easy inhalation.
6- S.typhi Immunization
The immunization program was performed as in flow fig.(1).
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Zainab et al. World Journal of Pharmaceutical Research
7- Blood Sampling
From each animal in each group 10 ml blood through cardiac puncture. Five with anticoagulant and five without anticoagulant. Those with anticoagulant was for cellular immune function, while, those without was fzor serology tests.
8-Immune Function Tests
Various immune function tests were done as in Steven 2010. Cytokine determination was performed as in manufacture instructions.
Group I
Group II
Group III
Group IV
Four sequential increasing tuberculin concentrations as 1st week 0.00005 IU
2nd week 0.0005 IU
3rd week 0.005 IU
4th week 0.05 IU Leave four days then the rabbits receives two weekly oral doses and two weekly oral doses and two weekly intravenous doses of S.typhi O vaccine leave one week,bleed
Four sequential increasing tuberculin concentrations to the same rabbit groups 1st week 0.00005 IU 2nd week 0.0005 IU 3rd week 0.005 IU 4th week 0.05 IU leave one week then bleed
S.typhi
control 5
rabbits 1st and 2nd
weeks oral 3rd and 4th
weeks intravenousl y ,one week leave then bleed.
Saline control received saline as in III programe.
Fig.(1) Immunization protocol
RESULTS
Cellular Immune Function (Table 1)
Nitroblue Tetrazolium Reduction NBT
The percentage of NBT phagocytosis were 43,58,63 and 31%for the groups I,II,III and IV respectively.
Leukocyte Inhibitory Factors(LIF)
LIF percentage were showing 74; 41.5; 52.03 and 98.8 for the groups I,II,III and IV respectively.
Erosette Count
The E.Rosette T-cell counts were showing percentage of 72; 71.5; 71.6 and 27.5 for the groups I,II,III and IV respectively.
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Zainab et al. World Journal of Pharmaceutical Research
III- Delayed Type Hypersensitivity skin test (table-3):
Using tuberculin and S.typhi cell free culture filtrate(CFCF) as skin sensitizer through ID rout to rabbits group I,II,III and IV. It was evident that four scores were noted as typical tuberculin , tuberculin type, Jones Mote Reaction and anergy.The induration area were 5,6,17 and 0 mm for the groups I,II,III and IV respectively.
IV- Humoral antibody responses (Table-4):
The anti S. typhi “O” agglutinin titers were showing 96,20,384 and 20 for the groups I,II,III and IV. The concentration of S.typhi IgM were 0.0816, 0, 0.512 and 0.073 while for S. typhi “O” IgG were 0.0298, 0, 0.231&0.017 for the groups I,II,III and IV respectively. IgM were of higher concentration than IgG in the groups I,III and IV respectively. Antituberculin antibody titer were showing 0, 80,0 and 0 in the groups I,II,III and IV respectively.
Table-1:Cellular Immune Feature of test and control rabbits.
Table-2: Cytokine profileand Cytokine unbalance
Feature
T est
Controls
T u+S. typhi
Tu
S.typhi
saline
NBT
43
58
63
31
LIF
74.5
61.5
52.03
98.8
E.Rosette
72
71.5
71.6
27.5
Cytokine
T est
Control
T u+S. typhi
Tu
S.typhi
saline
A-Profile
IL-1?
91.33
220.477
56.69
15.3
IL-12
60.454
24.477
24.56
7.32
IL-10
15.374
21.145
18.8
4.5
IL-4
2.4
1.811
0.07
99.1
B-Cytokine unbalance
Groups
Proinflammatory
Antinflammotory
IL-1?
IL-12
IL-4
IL-10
Tu+S. typhi
91.33
5.133
2.7
15.374
Tu
220.44
74.477
1.811
21.147
S.typhi
56.64
24.56
0.017
18.8
saline
75.3
7.32
99.1
4.5
.
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Zainab et al. World Journal of Pharmaceutical Research Table-3: Delayed hypersensitivity Skin Test in Test and Control Rabbits.
Table-4: Humoral Antibody Responses of Test and Control Rabbits
Group
Erythema 24-48hr
Induration 48-96hr
Jones Mote Reaction
CFCF
Tu
CFCF
Tu
CFCF
Tu
Tu+ S. typhi
++
+/++
6
5
1/5
2/5
Tu
+
0
0
0
-
0
S. typhi
++\+++
-/-
0
17
0
1/2
Saline
-
-
-
-
-
-
Type
Titer
Tu+ S. typhi
Tu
S. typhi
Saline
Anti- S. typhi
96
20
384
Antituberculin
0
80
0
0
IgM. antiS. typhi
0.0816
0
0.5125
0.073
IgG anti S. typhi
0.0298
0
0.231
0.017
DISCUSSION
The NBT neutrophil phagocytosis in the test and S.typhi control were higher than the saline control (Table-1). Group-1 although was showing increase as compared to saline control but it was lower than group II and III. There may be a competing epitope in S.typhi that might suppress NBT phagocytosis. LIF in group I and IV was non-significant but group I was around 70% and group II Tu alone poses significant low 40.5% LIF. Thus an epitope from S.typhi may inhibit LIF cytokine production in group I [10], [11]. CD2 is documented in rabbit lymphocyte of T-cell subsets. Control, saline showed 27.5% the other groups were showing counts around 70%, this mean that S. typhi , Tuberculin+ S. typhi and Tuberuculin Induce increase in T cell counts [12]-[15].The cytokine IL-4 was suppressed by treatment with S. typhi, Tuberculin+ S. typhi while tuberculin increase IL-1?, IL-12 and IL-10 in group II and to lesser extend in group I[16]. IL-1?, IL-12 as proinflamatory cytokines were highly exceeding IL-10 and IL-4 as anti-inflammatory cytokines a situation that present a case of cytokine unbalance (Fig-2).Single 1 IU in a dose of 0.1 ID yield 13-15mm induration area in tuberculin treatment rabbits one week post injection [17].
Chronic exposure to increasing low doses of tuberculin (Table-3) suppress the induration areas in treated rabbits group I. to the mean of 5 mm and to nil in group I. Thus such chronic exposure can be skin DTH suppressor, T cell competition (Tu+S.typhi) [18] or it present a case of low dose cellular tolerance[17],[19].
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Zainab et al. World Journal of Pharmaceutical Research
Jones Mott reaction was noted in 1/5, 2/5 of animal in group I and III but not in group II. With CFCF and Tuberculin as sensitizer in group I and III respectively [20]. Group I was showing low anti S. typhi “O” antibody titer as compared to group III. This can be due to competitor epitope in tuberculin that suppresses S. typhi “O” agglutinin titer[21]. S. typhiIgM index were far more than IgG in group I, and higher than group III and IV. Chronic exposure to tuberculin at low concentration inhibit class switching to IgG [2]. The cumulate tuberculin chronic exposure lead to increase of NBT, LIF, E.rosette, IL-1?, IL-12,IL-10 but not IL-4 while combine Tu+S. typhi exposure lead to NBT, E.rosette and IgM anti S. typhi . Both exposure modalities shift cytokine balance towards proinflamatory cytokines [22]. Therefore, tuberculin and tuberculo-typhoid exposure modulate rabbit immune system towards immunstimulation rather than immunsuppresion. (Table 1-4). Humoral and cellular antigenic competition were noted in S. typhi agglutinin and DTH induration respectively (table 3 and 4). Shift toward proinflamatory cytokines were noted (table 2) and inhibition of class switching from IgM to IgG (table 4).
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