World Journal of Pharmaceutical Research et al. World ... · Teaching Hospital. This study also involved twenty healthy persons as a control group. Patients group were subjected to

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Al-Jumaily et al. World Journal of Pharmaceutical Research

IMMUNOLOGICAL STUDY OF GASTRIC-ULCER PATIENTS

INFECTED WITH HELICOBACTER PYLORI

1Suha Talib Yahya Al-Jumaily, *

1Rajwa Hasen Essa, and

2Mohammed Issa Muhsin

1Department of Biology, College of Science, University of Mustansiriya.

2Immunology Dep. / Central public health laboratories

ABSTRACT

This study includes collection of biopsy and blood specimens from

patients with age ranged between 17-76 years that suffering from

dyspepsia, gastritis, duodenal ulcer, and gastric cancer during the

period between December 2011 to April 2012 at AL-Kadhymia

Teaching Hospital. This study also involved twenty healthy persons as

a control group. Patients group were subjected to gastroduodenal

endoscopy, gastric biopsy were taken from them for bacteriological

investigations. Blood samples were also collected for immunological

tests, while only blood samples were collected from control group. The

Detection of Helicobacter pylori was performed using several

laboratory investigations such as biopsy urease test, bacterial culture,

precipitation test, and serological test which include immunoglobulins and complement

quantitation and enzyme Linked Immunosorbent Assay (ELISA) for Interferon gamma (IFN-

γ) and Interleukin-8 (IL-8) detection. Outer membrane proteins were extracted from these

bacteria using Murphy method and the concentration of proteins were calculated by using

spectrophotometer, then proteins detected and the molecular weight of proteins were

evaluated by using electrophoresis. The stydy showed the prevalence of H. pylori in

(44.56%) of 92 patients. There was no significant relation between age factor and distribution

of H. pylori, but there is a significant relation between gender factor and distribution of H.

pylori. The result of biopsy culture was (7.31%) of (41) infected patients, while biopsy urease

test and Rapid anti H. pylori test gave (44.56% and 64.13%) respectively of 92 patients.

Electrophoresis results revealed ten bands which ranged between (16.914 – 340.476) KDa

with protein concentration of (2.76028) mg/ml. Outer membrane proteins were extracted

from H. pylori isolates and used as antigen to detect specific H. pylori antibodies using

World Journal of Pharmaceutical Research SJIF Impact Factor 5.045

Volume 4, Issue 1, 320-335. Research Article ISSN 2277– 7105

Article Received on

01 Nov 2014,

Revised on 25 Nov 2014,

Accepted on 20 Dec 2014

*Correspondence for

Author

Dr. Rajwa Hasen Essa

Department of Biology,

College of Science,

University of

Mustansiriya, Baghdad

100522.


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precipitation test in which the formation of precipitating line considered as a positive result.

High titer of IgG, IgA and C3 appeared in patients group, while a normal titers of IgM and

C4, as a result of immunoglobulins and complement quantitation. Enzyme-Linked

Immunosorbent Assay for detection of IFN-γ and IL-8 revealed statistically significant high

level of both in compared with control group.

KEY WORDS: Helicobacter pylori, Gastric, immunological aspects.

INTRODUCTION

The starting point of a revolution concerning the concepts and management of

gastroduodenal diseases is discovery of Helicobacter pylori which is well accepted, that is

most common stomach disease, peptic ulcer disease, is an infectious disease, and all

consensus conferences agree that the causative agent, H. pylori, must be treated with

antibiotics. Furthermore, the possibility emerged that this bacterium could be the trigger of

various malignant diseases of the stomach and it is a model for chronic bacterial infections

causing cancer.[1]

H. pylori colonize the human stomach, and then cause peptic ulceration, gastric lymphoma,

and gastric adenocarcinoma, the second leading cause of death from cancer worldwide. H.

pylori colonization occurs in childhood and persists throughout life, causing disease mainly

in adults. However, despite the fact that about half of the world’s population carries H. pylori,

only a small proportion develop ulcers or gastric cancer.Its spiral shape and flagella allow it

to corkscrew through the gastric mucus gel, and numerous adhesions enable selective

adherence to the epithelium. H. pylori has multiple mechanisms for protection against gastric

acid; notably, 15% of its protein content comprises preformed cytoplasmic urease. When the

external pH is less than 6.5, a specific channel opens in the bacterial cytoplasmic membrane,

allowing ingress of urea.[2]

Detection of antibodies specific to H. pylori in serum is an important in the diagnosis of this

bacteria. However, multiple invasive and non-invasive methods are available for the

detection of H. pylori. Invasive methods necessitate endoscopy and require gastric tissue.

They include tests for urease activity, histological evaluation, and culture of the bacterium.

Noninvasive techniques to detect bacterial infection include urea breath tests (UBT) and anti-

H. pylori antibody detection by serologic methods. Serologic tests offer high sensitivity and

specificity; furthermore, simultaneous measurement of serum immunoglobulin G (IgG), M


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(IgM), and A (IgA) antibodies to H. pylori can be used to determine the prevalence of both

acute and chronic infection. Serological tests are useful in H. pylori infection because

virtually all patients colonized with this bacteria undergo a local antibody response directed

against antigens covering the surface and flagella of the bacteria; in the majority of cases this

antibody response is also detectable in the serum.[3]

H. pylori possess a large outer membrane protein (Hop) family. It includes 32 membres, such

as adhesion protein, proinflammatory protein, and micropore protein. Although some

functions of these OMP have still been indefinite, the documents show that Hop is

significantly associated with high H. pylori colonization, the damage of gastric mucosa, high

mucosal IL-8 levels, and neutrophil infiltration.[4]

Several porins of OMP are also

immunologically active and can act as protective antigens, and together with the LPS they

often represent the most significant antigenic determinants of a particular bacterial species.[5]

For the previous description of the importance of H. pylori infections and diagnosis by using

bacterial Ag and specific Ab, this study aimed to: shoot light on H. pylori prevalence,

determine some humoral immune response factors (IgM, IgG, IgA), detect the interleukins in

patients sera, study OMP and use it as potent specific Ag for H. pylori detection.

MATERIALS AND METHODS

Specimens' Collection

The specimens were collected under consultation of physician medicine consultant during the

period between December 2011 and April 2012 at AL-Kadhymia teaching hospital in

Baghdad city.

Antral Biopsy Specimens

One gastric antral biopsy specimen was taken from 92 patients with, dyspepsia, gastritis,

duodenal ulcer and gastric cancer, the age was ranged between 17 – 76 years. Biopsy were

collected in sterilized test tube contained urea broth medium for urease activity test (RUT)

and for transporting the biopsies to the laboratory for cultivation in cool box at 4ºC for not

longer than 4 hours before processing. Biopsy forceps were washed with water and

disinfected with glutaralde- hyde (cidex) for 10 min and then washed with sterilized distilled

water also before and after each procedure.[6]


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Blood Samples

Five mL of blood were collected from 92 patients plus 20 control, the age ranged between

17–76 years.The blood samples were divided into two parts: 1 mL in heparinized tubs for

rapid anti H. pylori test by use one Step H. pylori test card kit, and 4 mL in dry tubs for

serology. After clotting, the sera were obtained by centrifugation (for 10 min at 3000 rpm)

divided into aliquots and stored at (-20°C) until used.

Outer membrane proteins (OMP) Extraction and Partial Purification

Extraction of outer membrane proteins from H. pylori outer surface was carried out according

to Murphy et al.[7]

Estimation of Outer membrane proteins concentration: Proteins concentration was

determined according to Kalcker,[8]

by using the equation:

Concentration of = 280nm absorption × 1.55 - 260nm absorption × 0.76

protein (mg/cm3)

Detection of Molecular Weight using Sodium Dodecyl Sulphate – Poly Acrylamide Gel

Electrophoresis[9]

Resolving gel (10%) was poured in electrophoresis tubes, and these tubes were then left for

30 minutes to ensure complete solidification. Staking gel (3%) was added to resolving gel in

tubes, the tubes were then left for 15 mins for completing polymerization. After 24 hr, they

were placed in electrophoresis unit. The electrophoresis system was connected to the power

supply with current density 2mA/tube for 30 mins to remove positive ions for free protein

movement. The M.W of obtained band of OMP was compared with marker proteins of know

M. W.

Rapid Anti H. pylori Test (For Blood)[10]

Rapid anti H. pylori test was carried out according to the protocol of One Step H. pylori Test

Card Kit as follows: one mL of blood was taken from each patient and put in heparinized

tube. One drop of heparinized blood was taken and applied to the sample well of provided

strip in the Kit. Two drops of provided sample diluents were added. Result of the test was

read after 15-20 minutes.


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Reading the Test Result

Positive

Both purplish red test band and purplish red control band appear on the membrane.

Negative

Only the purplish red control band appears on the membrane. The absence of a test band

indicates a negative result. Invalid: There should always be a purplish red control band in the

control region regardless of test result. If control band is not seen, the test is considered

invalid.

Immunoglobulins and Complement Components Determination by Single Radial

Immunodiffusion Test[11]

Singles Radial Immunodiffusion (SRID) test were used for the quantitative determination of

many human serum proteins such as IgA, IgG, IgM, and C3, C4 complements. The procedure

consists in an immuno precipitation in agarose between an antigen and its homologous

antibody. It is performed by incorporating one of the two immune reactants (antibody) into

wells duly punched in the gel. Antibody diffuses radially out of the well into the surrounding

gel-antigen mixture, and a visible ring of precipitation forms where the antigen and antibody

reacted, Ring diameters are measured by hand lens (0.1mm precision) then concentration

were determined from the tables.[12]

Interferon Gama (IFN-) Determination

Interferon Gama (IFN-) Determination was carried out according to the protocol of Human

IFN- ELISA Kit.[13]

Method Principle

Human IFN- ELISA (Enzyme-Linked Immunosorbent Assay) kit was used for the

quantitative measurement of human IFN- in serum; this assay employs an antibody specific

for human IFN- coated on a 96-well plate. Standards and samples are pipetted into the wells

and IFN- present in a sample is bound to the wells by the immobilized antibody. The wells

are washed and biotinylated anti-human IFN-_ antibody is added. After washing away

unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The

wells are again washed, a TMB substrate solution is added to the wells and colour develops

in proportion to the amount of IFN- bound. The Stop Solution changes the colour from blue

to yellow, and the intensity of the color is measured at 450 nm.


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Interlukin-8 (IL-8) Determination: Interlukin-8 (IL-8) Determination was carried out

according to the protocol of Human (IL-8) ELISA Kit.[14]

Method Principle

The IL-8 EASIA is a solid phase Enzyme Amplified Sensitivity Immunoassay performed on

microtiterplate. The assay uses monoclonal antibodies (MAbs) directed against distinct

epitopes of IL-8. Calibrators and samples react with the capture monoclonal antibody (MAb

1) coated on microtiter well and with a monoclonal antibody (MAb 2) labeled with

horseradish peroxidase (HRP). After incubation period allowing the formation of a sandwich:

coated MAb 1 – human IL-8 – MAb 2 – HRP, the microtiterplate is washed to remove

unbound enzyme labelled antibody. Bound enzyme – labelled antibody is measured through a

chromogenic reaction. Chromogenic solution (TMB) is added and incubated. The reaction is

stopped with the addition of stop solution and the microtiterplate is then read at the

appropriate wavelength. The amount of substrate turnover is determined colourimetrically by

measuring the absorbance, which is proportional to the IL-8 concentration. A calibration

curve is plotted and IL-8 concentration in samples is determined by interpolation from the

calibration curve. The use of the EASIA reader (linearity up to 3 OD units) and a

sophisticated data reduction method (polychromatic data reduction) result in a high

sensitivity in the low range and in an extended calibration range.

Precipitation Test

Precipitation test was carried out according to Ochterlony test for Immunodiffusion in

Agarose Gel according to[15]

as follow: The patients sera were diluted 3 times as double

dilution. Six wells were made in agarose medium according to dilutions numbers of serum, in

this study, one well was made in the middle and five wells were made around it at the same

distance between them (5-7 mm). Twenty five µl of Antigen (Outer membrane proteins

extract) was added into the middle well while 25 µL of concentrated and diluent sera were

added into four wells respectively, and 25 µL of phosphate buffer saline was added into the

fifth well which considered as control. The plates were puts into a Desiccator jar with a pad

of cotton which was soaked in water and placed at the bottom to provide a humidified

conditions, and incubated at 37°C, then examined after 24 hr to 7 days. The appearance of

precipitation line between the Ag and Ab (serum) gave a positive result.


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Statistical Analysis

The Statistical Analysis System- SAS (2010) was used to effect of difference factors in study

parameters. The least significant difference (LSD) test at the comparative between means and

chi-square test to significant compare between percentage in this study.

RESULTS AND DISCUSSION

Clinical Status of Patients Group

As shown in table (1) endoscopically gastritis was diagnosed in (42.4%) of the total patients,

duodenal ulcer was seen in (25%), while (7.6%) of the patients had gastric ulcer, and (6.5%)

of the patients were presented with duodenitis, in other hand (7.6%) had both gastritis and

duodenitis. Non ulcer dyspepsia was also diagnosed in (3.3%) and (5.4%) had reflex

esophagitis. Gastric cancer was endoscopically diagnosed in (2.2%) of the patients, all these

cases were diagnosed under consultation of medical physicians, these results approached

those of Al-Dhaher.[16]

Table (1) Clinical State of Patients Diagnosed by endoscopy.

Clinical State Patients No. Patients Percentage

Gastritis 39 42.4

D.U 23 25

Gastric ulcer 7 7.6

Duodenitis 6 6.5

Gastritis and Duodenitis 7 7.6

Non ulcer dyspepsia 3 3.3

Reflex esophagitis 5 5.4

Gastric cancer 2 2.2

Total 92 100%

Determination of H. pylori in Patients

The presence of H. pylori was determined by biopsy urease test, rapid anti H. pylori test and

culture of antral biopsy specimens in 92 patients. As shown in Table (2) fourty one (44.56%)

patients were positive in biopsy urease test and fifty nine (64.13%) were positive in rapid anti

H. pylori test, according to this 41 biopsy were took from patients gave positive results in

both tests above, hence three (7.31%) patients were positive in culture. Patients were

considered to be infected with H. pylori if they were positive in two of the three test, (biopsy

urease test, rapid anti H. Pylori and culture). According to these results fourty one (44.56%)

patients were considered to be infected in this study. The use of multiple diagnostic methods

is recommended to accurately diagnose H. pylori infection.[16]

These results agree with the


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results of Al-Nami,[17]

who found the prevalence of H. pylori (48.23%) in Iraqi patients,

while disagree with the results of Al-Hadi,[18]

who found the prevalence of bacteria (74.1%).

Table (2) Diagnostic Tests for H. pylori Infection in 92 Dyspeptic Patients.

Tests Cases Total cases % +ve

Patients case +ve cases -ve cases

Urease 41 51 92 44.56

Rapid anti H. pylori 59 33 92 64.13

Culture 3 38 41 7.31

Distribution of H. pylori Infection According to Age Group

The data resulting from this study as illustrated in Table (3) shows high prevalence of H.

pylori infection in the age group ranging between (30-39) and (40-49) which reached to

11(26.83%) and 10 (24.39%) respectively, in spite of this, there was no statistically

significant differences when compared with control group and non-infected patients group at

p value (0.01). This is applied on the other age range, which explaned that there is no

relation between the age and the prevalence of H. pylori. Results of this study agree with the

results of Meng et al.[19]

and Al-Nami,[17]

who found that there is no important difference in

H. pylori prevalence between patients and healthy at different age range, and disagree with

the study of Al-Dhaher,[16]

which showed an extremely high prevalence of H. pylori infection

in the age group ranging from 10 to 19 years and the age 60 years. The differences between

the results may due to some factors as O blood group, crowd, education and smoking.[20]

Table (3) Number & presentage distribution of sample study according to Age.

Age group

Patients group Total of

patients group

Control

Group Infected Non Infected

No. % No. % No. %

10 – 19 1 2.44 2 3.92 3 1 5.00

20 – 29 8 19.51 10 19.61 18 5 25.00

30 – 39 11 26.83 13 25.49 24 4 20.00

40 – 49 10 24.39 8 15.69 18 3 15.00

50 – 59 4 9.76 10 19.61 14 3 15.00

60 – 69 5 12.20 6 11.76 11 3 15.00

70 – 79 2 4.88 2 3.92 4 1 5.00

Total 41 100% 51 100% 92 20 100%

Chi-square

value

6.844** 6.912** 6.402**

** (P<0.01), Non-significant


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Distribution of H. pylori Infection According to Gender

As shown in Table (4) 16 (39.02) from 37 males and 25 (60.98) from 55 females presented in

this study were infected. Statistically there are significant differences at p0.05 between

males and females. Results of this study confirmed that the gender is an effective factor in H.

pylori infection which has agreed with the results of Yass,[21]

in the ratio of infected females

(55.6%) was higher than ratio of infected males and disagrees with Baaker,[22]

and Al-

Nami,[17]

when explained that the gender is not considered as a risk factor in H. pylori

infection, also has disagreed with other studies[23,24]

which confirm the dominance of males

on females, that is many of males were smoker and alcoholic. Other studies confirm

approximately equality between the ratio of male to female which was (72.50%) to (75.3%)

respectively.[25]

Table (4) Number and percentage distribution of sample study according to Gender.

Gender

Patients Group Control Group

Infected Non Infected

No. % No. % No. %

Male 16 (39.02) 21 (41.18) 10 (50.00)

Female 25 (60.98) 30 (58.9) 10 (50.00)

Total 41 100% 51 100% 20 100%

Chi-square

value

9.554** 4.893* 0.00S

*(P0.05), **(P0.01), S: significant

Laboratory Investigation of Biopsy specimens

Biopsy Urese Test: The ability of H. pylori to produce urease enzyme allows one to rapidly

detect the organism in gastric biopsy material.[26]

In this test (44.56%) 41patients gave

positive results as shown in table (2). The test was read as positive when a pink or purple

color developed around the biopsy specimen in the inoculated media. Some of the cases

showed positive results after one hour and others after four hours, while others gave positive

results after an overnight incubation. There is a correlation between the number of bacteria

present per biopsy and the time for a positive reaction to appear. The more organisms seen in

the biopsy the more likely the biopsy urease test will be positive within a short time.[16]

In this

study 18 patients were negative in biopsy urease test but they were positive in rapid anti H.

pylori test, the negative results may be explained as follows: At least 105 per ml bacteria are

needed for a positive result and the use of two biopsy specimens increase the sensitivity of

the test.[27]

Performed enzymes cause the reaction; therefore sample size might affect


329


reproducibility.[28]

This may also due to the irregular distribution of the organism in the

gastric mucosa (patchy).

Culture

H. pylori was cultured from 3 out of the 41 infected patients. Isolation and identification of

the organism by culture approximately took (5-7) days. Growing bacteria were identified as

H. pylori based on the morphology of the colonies, gram staining, oxidase, catalase, urease

production.[25]

Growing colonies identified in this study were small translucent like water

spray. The organism was gram negative, curved or bacilli and it was catalase and oxidase

positive also it has a characteristic urease most of positive cases became positive in urease

test within 15 minutes while a few of them became positive within 30-40 minutes and this

agrees with the result of Al-Rawi,[29]

who found some isolates gave positive results within 30

minutes while disagrees with the result of Mohammed[30]

who found positive results given

within 2-3 hours. The remaining 39 infected patients, failed to culture due to the fastidious

nature of H. pylori or because of the patient had taken antibiotic and because of a number of

other factors like the patchy distribution of the organism, inadequate mincing of the biopsy

material, the presence of oropharyngeal flora, or the loss of viability of the specimen during

transportation.[31]

Culture has not proved to be sensitive unless multiple biopsy specimens

collected from the gastric mucosa are processed. This is due to the heterogeneous distribution

of organisms (Murray et al., 1994). The percentage of culture was 3 (7.31%) of 41 infected

patients which agree with the result of Kaore et al.[31]

who found the percentage of culture

was (8.69% ).

Outer Membrane Protein (OMP) Extraction

One isolate was selected for outer membrane proteins extraction which was made by using

sonicater which refract most of the cell according to Murphy et al.[7]

to be sure that the cells

were refracted, smear was made and simply stained. The addition of DNase, RNase enzymes

was to decrease viscosity of solution through refraction of DNA, RNA moleculs, the

Lsozyme split Murine and weaken linkage between peptidoglycan layer and outer membrane

proteins and then make the exposure to SDS detergent greater. SDS detergent does not

dissolve Outer membrane proteins but selectively dissolve Inner membrane proteins.[32]

Evaluation of Protein Concentration in the OMP Extraction

According to Kalcker, (8) the solution absorption evaluated at 260nm and 280nm before and

after dialysis. The final protein concentration was (2.76028) mg/mL.


330


Partialy Purified and Detection of Protein by Sodium Dodecyl Sulphate–Poly

Acrylamide Gel Electrophoresis

H. pylori outer membrane proteins were analyzed by 10% SDS-PAGE and the lane of protein

bands obtained were compared with six marker proteins (Esterase MW=230KDa, ɣ-globulin

MW=150KDa, Transferrin MW=80KDa, Bovine serum albumen MW=67KDa, Trypsin

MW= 23KDa, Lysozyme MW=14KDa) Table (3-4). Results of protein profile by SDS-

PAGE revealed ten bands with M.W ranged between 340.476 – 16.914 kDa Table (5), (6).

Table (5) Molecular weight of standard proteins.

Standard proteins M. W. kDa

Esterase 230.000

ɣ-globulin 150.000

Transferrin 80.000

Bovine serum albumen 67.000

Trypsin 23.000

Lysozyme 14.000

Table (6) Molecular weight of Helicobacter pylori proteins.

Number of proteins M. W. kDa

1 340.476

2 298.571

3 189.697

4 145.069

5 112.091

6 92.559

7 79.024

8 73.198

9 67.000

10 16.914

Laboratory Investigation of Blood and Serum Specimens

Rapid Anti H. pylori Test

This test is a simple, visual qualitative test that detects antibodies in human whole blood,

serum or plasma. The test is screening test and can be use in the laboratory as a routine test. It

is based on immunochromatography and can give result within 15 minutes.

Determination of Immunoglobulins and Complement Components

Quantitation of immunoglobulins (IgG, IgA, IgM) and complement factors (C3, C4) provide

useful information for the evaluation of certain disease states. As shown in table (7) the

means of IgG, IgA, titers showed high levels compared with the normal values, the statistical


331


analysis showed that there are significant differences between patients and control in IgG and

IgA titers. But normal titers of IgM were seen in patients group and statistically there were no

significant differences between patients group and control group in IgM titer.

Serrano et al. reported a significant elevation of IgG and IgA titer in H. pylori patients serum,

but IgM does not present an additional contribution.[33]

While She et al. explain that IgM has

been found to have little diagnostic utility for H. pylori infections and is elevated only acutely

after infection, whereas H. pylori infections are generally chronic, that is IgM has extremely

low sensitivity.[34]

Table (7) Comparson among the mean of concentration of Immunoglobulins isotypes in

patients serum (IgA, IgG and IgM) and control group.

Group Isotypes of Immunoglobulins in serum

IgA (mg/dl) IgG (mg/dl) IgM (mg/dl)

Patients 284.13 ± 22.07 1208.42 ± 126.99 139.69 ± 7.57

Control 137.67 ± 13.61 546.53 ± 63.14 74.81 ± 8.59

LSD Value 80.281 * 208.81 * 29.003 *

* (P<0.05).

While Table (8) shows an increasing in C3 levels in patients group and statistical analysis

showed significant differences between patients group and control group. Complement

activity is essential for the bactericidal effect. This is in line with the observation by Desar

et al. that is H. pylori is complement-sensitive and activates the classic pathway even in the

absence of specific antibodies.[35]

Normal titer of C4 were seen in patients group and

statistically there are no significant differences between patients group and control group.

Table (8) The mean concentration of C3 and C4 value of patients and control group.

Group Complement Component

C3 (mg/dl)

Mean ± SE

C4 (mg/dl)

Mean ± SE

Patients 143.46 ± 6.68 30.12 ± 1.52

Control 65.74 ± 9.94 18.82 ± 1.91

LSD Value 26.506 * 5.893 *

* (P<0.05).

Interferon Gamma (IFN-γ) and Interleukin-8 (IL-8) Detection

Levels of numerous cytokines, including interferon gamma (IFN-γ) and IL-8, are increased in

the stomachs of H. pylori-infected humans compared to uninfected humans.Table (9) shows a


332


high level of IFN-γ and IL-8 in patients group as compared with control group, and statistical

analysis give significant differences between patients and control groups at p<0.05.

Table (9): The mean concentration of IL-8 and INF- γ in serum of patients and control

group.

Group Mean ± SE

IL-8 (pg/ml) INF- γ (pg/ml)

Patients 0.7167 ± 0.06 297.44 ± 22.57

Control 0.446 ± 0.07 1.32 ± 0.15

LSD Value 0.267 * 65.345 *

* (P<0.05).

Study of Bimczok et al. showed that H. pylori induced substantially higher levels of IFN-γ

and IL-8. In humans, IFN-γ sustains mucosal inflammation and may promote disease

progression to gastric ulcer.[36]

Another study on mice by Zhang et al. showed an increased

levels of IFN-γ in the stomachs of H. pylori-infected mice is consistent with the development

of a Th1-predominant response,[37]

and other study by Peek et al. reported that IL-8 is

increased within H. pylori-infected mucosa where it localizes to gastric epithelial cells, and

levels of IL-8 are directly related to the severity of gastritis.[38]

Detection of Antibodies for H. pylori OMP

Precipitation Test

H. pylori OMP extract was used for antibodies detection using Ouchterlony gel diffusion

test, the result showed forming of precipitation line between Ag (partial purified OMP) and

Ab (patients antisera) at dilution 1:4 while not forming any other precipitation line at the

other dilution (1:2,1:8). This result means that the Ag was high specific against anti H. Pylori

antisera[15]

and can be used to detect H. Pylori antibodies in patients sera, so the following

primary results encorouge us for additional studying about OMP.Precipitation line depends

on the diffusion rate of the Ag and its purity hence our results might indicate the purity of

OMP in addition to its specificity.

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World Journal of Pharmaceutical Research et al. World ... · Teaching Hospital. This study also involved twenty healthy persons as a control group. Patients group were subjected to

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