Journal of Babylon University/Pure and Applied Sciences/ No.(8)/ Vol.(21): 2013
Lapin Mucosal Humoral and Cellular Versus Systemic
Humoral and Cellular Immune Responses Post To
Rectal Administration
Of Heat Killed Helicobacter Pylori Bacterin
Qasim N.A. Thewaini
Ala’a J. Hassan
Babylon University , College of Science , Department of Biology
Samah A. Kadum
Babylon University , College of Pharmacy , Department of Biology.
Abstract
Rapid non-invasive diagnostic tests that can reliably document the presence or absence of
Helicobacter pylori infection are urgently required. Four successive dose of heat killed H.pylori
(HpHK) bacterin was rectal administration in rabbits at five days a part manner.
HpHK bacterin stimulate specific mucosal and systemic humoral and cellular immune responses by
increased immunoglobulins titers and concentrations, increased the concentrations of C3 and C4 which
are the complement compartments with significant increased in the lymphoid cells that formed the
rosette form , significant migration inhibition index , increased the concentrations of cytokines . HpHk
induced positive skin test tuberculin type delayed hypersensitivity, in addition to the histopathological
changes at different body organ portions of the animals.
‫الخالصة‬
‫إن استعمال االختباا اا اتتخخصياصغ ر اا ااتصا صاغ اتساا عغ صعطا كرااع ان اأو دو اأث اأو ابياابغ االوماغ ات وت صاغ‬
‫م صاغ‬
‫ ااى ت ض ا برتا ن مقتول بات اااع من هذه اتاالومغ وتاث تاا عال تا اا ان ان طا اخ اتمخااع باعاوع ااات و ام كتا اا‬. ‫ات وا صغ‬
‫ فا ا ه ااذا اتبرت ااا ن اس ااتاابغ م ا ص ااغ اةا ااغ وموض ااعصغ خ طص ااغ وخ وص ااغ م اان خا ا ل ااتف ااات مع ااأل تار ا ا و ص اااا ا ض ااأاأ‬. ‫مخت ف ااغ‬
‫ااأأ اتخ صاا ات مفصااغ اتمرو ااغ ت تخاار ات هااا‬
‫ات ارصاا‬
‫بابضاااكغ إتاام‬
‫اااأع كا‬
‫ كا اتميا مااع اأو‬C4 ‫ و‬C3 ‫اتماتمث‬
‫كا ا و صاغ اتخاعا غ وااوأ االااا ن اتم فا بابضااكغ إتام االاتفاات كا تار ا بعا‬
‫ما اااا ن ص فا كاااي ات ساسااصغ اتمتااعخا كا اتا ااأ ماان ات ااوت اتت ااووار‬
. ‫ضاء اتمخت فغ اتمعخوذع من د اا ن االختباا‬
‫اتمتخيياغ وااتفااات تار ا مرااو‬
‫و أو تل صط ك هااع اتخ صا ات اص‬
‫ رمااا تااو ذ إن هااذا اتبرتااا ن صعما‬. ‫اتخ وصااغ‬
‫اتماضصغ ات ساصغ ك ا‬
‫أو اتتغ اا‬
Introduction
Host immune responses have been linked to susceptibility to gastroduodenal
diseases in Helicobacter pylori infection. Most data suggest that a mucosal and
systemic T helper type 1 (Th1) response is associated with peptic ulceration , but the
role of Th2 cells is less clear (David etal., 2004). Helicobacter pylori has been well
established that infection with this gastric pathogen leads to chronic–active gastritis,
and may be complicated by peptic ulceration both in adults and in children. (Andrew
etal., 2002) . Rapid determination of the presence of anti- H. pylori antibodies in
clinical practice settings would assist in defining appropriate further investigations
and management steps in the evaluation of patients with possible H. pylori infection.
It cause mucosal as well as systemic humoral & cellular immune responses , till now
little references regarding the immune status of the lapin animal primed with this
bacteria and the time of development of these responses (Kuster etal., 2006 ;
Aguemon etal., 2004).
2764
Objectives
The aim of this study is the compare between mucosal and systemic humoral
and cellular immune responses in rabbits after rectal administration of heat killed
H.pylori (HpHK) bacterin at different days.
Materials & Methods
1- Antigens:
Heat killed H.pylori (HPHK) bacterin was prepared from 24 hour brain heart
infusion agar plate culture then we add 6 ml of normal saline to the plat’s scrape ,
collect the solution and put it in centrifuge at 4000 rm\ mint for 5 mints, double wash
were done with normal saline then compared it with standard opacimeter (WHO) to
obtain the concentration equals to 10 IU\ml . The suspension tubes were used after put
them in water path at 60 degrees for 30 minute to killed the bacteria and obtained the
antigen that was used in immunization the rabbits after done the sterility test . A cell
free culture filtrate antigen was also prepared from microaerophilic 72 hour brain
heart infusion broth culture of H.pylori (Svanborg-Eden etal., 1985 and Sachse etal.,
2005) .
2- Animals:
Two groups, each of two rabbits Oryctolagus caniculus were elected , adapted
to laboratory conditions and housed under Ad libitum standardized conditions , one
served as test & other as control group (Schneider etal., 1990).
3- Immunization protocol :
Four successive doses of (HpHK) bacterin were administered via rectal rout into
tested rabbits through four weeks , each dose about 2 ml of bacterin that had 10
IU\ml concentration . Control animals received sterile normal saline in same protocol
and this protocol was specific for this research.
4- Mucosal samples and immunoglobulins separation :
Gut mucosal samples were obtained from three parts of gut mucosa included
esophageous , stomach and duodenum in addition to spleen which is used as lymphoid
organ. Then the immunoglobulins were separated from these parts according to
Shnawa and Abid(2005). .
5- Blood Samples
Blood with anticoagulant was processed for E-rosette test and for migration
inhibition test. Coagulated blood were collected for the others immunological tests
that include: tube agglutination test , measure the concentrations of IgG , IgM ; C3 ,
C4 ; IL-4 and IL-8 (Garvey etal., 1977)
6-Immunology:
Agglutination test was performed by microtiteration method, anti H.pylori
the IgG, IgM antibodies were detected by using specialized kits were provided from
(DRG, USA) company . The C3 and C4 components were determined by C3 and C4
proteins , E-rosette , Capillary Migration Inhibition were performed as in (Garvey
etal., 1977), IL-4 and IL-8 cytokines were assayed using ELISA kits ( provided from
RayBio, USA, Company), skin delayed type hypersensitivity was done as in (Shnawa
and Abid, 2005). Histopathology was carried out as in (Kuster etal., 2006)
Results and Discussion
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Journal of Babylon University/Pure and Applied Sciences/ No.(8)/ Vol.(21): 2013
H.pylori possess several immunogenic subfractions , such immunogens
stimulate B cells , T cells as well as the subset Tdh responsible for hypersensitivity
(Velin etal., 2004 and Choi etal., 2011) . The gut compartment, the common mucosal
immune system was attempted as a model for providing the immunological opinion
that mucosal immunization induces mucosal as well as systemic immune responses
specific to the stimulating antigen (Ferrero, 2005 and Michetti, 2011 ). We studied
the development of immune responses at five days (8 , 12 , 16 , 20 and 27) after
immunization with HPHK bacterin via rectal route that stimulates H.pylori specific
humoral systemic and mucosal immunoglobulins titers (Table 1) , also it played an
important role in the increased the concentrations of IgG and IgM anti H. pylori
antibodies in serum and mucosal secretions of immunized rabbits
(Table 2). The
concentration of C3 and C4 which are the complement compartments were
significantly increased in the sera of rabbits (Table 3) (Ferrero etal., 2005 ; Sutton and
Mitchell, 2010).
The cellular systemic and mucosal immune responses were represented by
significant increased in the lymphoid cells that formed the rosette form , significant
migration inhibition index with more than 30% inhibition as well (Table 4),
furthermore, the HPHK bacterin able to inducing tuberculin type delayed
hypersensitivity. Hence, their epitopes can be of T dependent type through the
activation of Th1 and Th2 (Table 5) (Kayaselcuk etal., 2002 ; Velin etal., 2004 and
Michetti, 2011).
IL-4 is one of anti-inflammatory cytokines and IL-8 is a proinflammatory
cytokine were be detected in sera and mucosal secretions of rabbits with significant
increased in their concentrations (Table 6) . Thus H.pylori antigens were B and T cells
dependent types (Svennerholm and Lundgren, 2007). The histopathological study
was showed the splenic reactive hyperplasia in red pulp in the days 8 and 12
respectively, in addition to the presence of large number of lymphocytes that
converted to the plasmocytes, with the appearance of the case of immune tissue
injury in lymphoid follicles (picture 1), dearrangement of epithelial cells that lining
the mucosal layer of esophageous (Picture 2), increased the surface area of the pits of
mucosal layer of rabbits stomach, also we have seen the infiltration of neutrophils to
the lamina propria of the stomach (Picture 3), while the duodenum had been normal
in all treated rabbits. Rectal compartment is being induction compartment of mucosal
immune system . HpHK induce both humoral and cellular
immune responses both at mucosal and systemic levels.
Titer
Types of immune response
Days
8
12
16
Control
20
Table (1): Titers of anti H.pylori antibodies
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27
Humoral systemic(serum)
Cellular mucosal
Esophageous
Stomach
Duodenum
20
160
320
640
1280
10
8
16
16
16
32
32
32
64
128
32
128
256
64
128
256
1
Table (2) : Concentrations of anti-IgG and anti-IgM H.pylori antibodies
Types of immune
response
8
4.036
±
0.048
Humoral
systemic(serum)
Cellular mucosal
Esophageous
3.724
±
0.003
3.930
±
0.007
3.722
±
0.006
Stomach
Duodenum
Control
IgG concentration(Iu\ml)
M±S.D.
Days
12
16
20
4.241
4.447
4.450
±
±
±
0.008
0.000
0.017
3.931
±
0.014
4.137
±
0.009
3.723
±
0.002
4.136
±
0.106
4.766
±
0.004
4.136
±
0.003
4.076
±
0.036
4.140
±
0.049
5.560
±
0.001
4.137
±
0.006
27
4.549
±
0.002
IgM concentration(Iu\ml)
M±S.D.
Days
8
12
16
20
3.335
3.613
3.813
3.975
±
±
±
±
0.004
0.015
0.006
0.004
27
3.813
±
0.098
4.140
±
0.005
7.944
±
0.018
4.342
±
0.002
3.019
±
0.002
3.490
±
0.000
3.336
±
0.004
3.973
±
0.093
4.342
±
0.002
3.975
±
0.097
3.177
±
0.000
3.815
±
0.004
3.336
±
0.002
3.975
±
0.025
4.548
±
0.001
3.973
±
0.149
3.638
±
0.001
Table (3) : Concentrations of C3 and C4 in the sera of rabbits
Days
C3 concentration (mg\dc)
M±S.D.
C4 concentration (mg\dc)
M±S.D.
8
108.450
±
2.899
147.100
±
9.616
205.850
±
3.747
255.400
±
4.101
285.000
±
4.242
36.500
±
0.989
47.950
±
1.060
57.300
±
1.131
80.050
±
1.343
81.000
±
2.687
12
16
20
27
2767
4.131
±
0.113
4.549
±
0.000
4.131
±
0.172
Journal of Babylon University/Pure and Applied Sciences/ No.(8)/ Vol.(21): 2013
Control
110.500
±
0.000
37.200
±
0.000
Table(4) : Percentage of E-rosette and LIF tests
Types of
immune
response
Cellular
systemic
Cellular
mucosal
Esophage
ous
Stomach
Duodenu
m
Control
8
28.750
±
0.494
Percentage of E-rosette(%)
M±S.D.
Days
12
16
20
30.250
31.450
33.450
±
±
±
0.070
0.636
0.636
29.700
30.850
34.300
±
±
±
0.707
0.070
0.989
30.100
33.700
39.300
± of inoculation
±
±
Date
0.565
0.848
0.848
CFC \ Days
30.850 635.250
39.150
±
±
±
2.687
0.070
0.636
10
27.500
±
0.427
14
18
27
34.700
±
0.424
8
90.700
±
0.707
34.200
35.800
89.400
±
±
±
0.282
0.141
1.272
34.850
36.700
76.950
Skin±test \ Hours
±
±
6 1.060
18
24
48
0.000
1.202
41.750
42.600
79.000
E
EI
EI
±
±
±
10
10
1.979
0.424
1.131
E
EI
EIN
15
15
E
EI
EI
8
8
E
EI
EI
Table (5) : Skin test
2768
Percentage of LIF(%)
M±S.D.
Days
12
16
20
89.150
85.050
75.250
±
±
±
0.070
0.141
0.424
87.150
±
0.494
73.100
±
72
0.282
70.300
EI
±
10
1.414
EIN
15
EI
8
EI
27
65.850
±
0.777
80.100
72.850
63.900
±
±
±
0.707
0.353
0.636
70.650
68.050
63.150
±
Immune ±reaction ±
0.777
1.343
0.919
70.000 Notes
63.950
59.450
±
±
±
Reaction0.636
area (mm)
1.131
0.636
93.500 Notes
±
Reaction
area (mm)
0.427
Notes
Reaction area (mm)
Notes
-
25
Control
-
Note : E- Erythema
5
-
5
-
I- Induration
5
-
Reaction area (mm)
Notes
Reaction area (mm)
Notes
Reaction area (mm)
N- Necrosis
Table (6) : Concentrations of IL-4 and IL-8
Types of
immune
response
Cellular
systemic
Cellular
mucosal
Esophageous
Stomach
Duodenum
Control
8
7.248
±
0.070
IL-4 concentration(pg\ml)
M±S.D.
Days
12
16
20
11.116
149.676 151.124
±
±
±
0.213
0.963
0.234
6.284
±
0.042
4.836
±
0.065
6.280
±
0.203
6.696
±
0.048
7.000
±
0.148
7.176
±
0.332
7.080
±
0.106
7.720
±
0.514
7.256
±
0.135
7.004
±
0.012
9.176
±
0.087
9.176
±
0.526
9.664
±
0.152
27
156.436
±
0.678
8
18.448
±
0.462
IL-8 concentration(pg\ml)
M±S.D.
Days
12
16
20
18.844
20.124
20.508
±
±
±
0.384
0.329
0.401
6.764
±
0.374
7.936
±
0.076
7.040
±
0.055
18.252
±
0.278
14.964
±
0.111
18.276
±
0.205
19.596
±
0.745
19.884
±
0.045
21.552
±
0.202
A
B
2769
21.564
±
0.127
20.352
±
0.384
21.564
±
0.364
19.814
±
0.050
23.220
±
0.613
23.232
±
0.451
26.508
±
0.708
27
20.892
±
0.004
26.532
±
0.274
38.136
±
0.028
26.532
±
0.253
Journal of Babylon University/Pure and Applied Sciences/ No.(8)/ Vol.(21): 2013
plasmocyte
C
D
Immune tissue injury
Immune tissue injury
E
F
Picture (1): Section in spleen
A- control group , B- & C- splenic reactive hyperplasia , D- plasmocyte ,
E- & F- immune tissue injury in lymphoid follicles (100X, 400X, 1000X)
A
B
Picture (2): Section in Esophageous
A- Test group , B- Control group (400X)
2770
A
B
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Picture (3): Section in Stomach
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