Determining the IgM and IgG antibody titer against CMV and helicobacter pylori in the serum of multiple sclerosis patients comparing to the control group in Hamadan

Abstract

BACKGROUND:

Multiple sclerosis (MS) is a chronic autoimmune disease that disables central nervous system (CNS) system. Cytomegalovirus (CMV) probably has an important role in the MS pathology. The infection with helicobacter pylori also is recognized as a protective agent against MS in female.

METHODS:

Serum samples were isolated and frozen at $-$ 70 ${}^{\circ}$ C. The earlier mentioned anti-virus antibodies and antibacterial antibodies were quantified by Elisa kit.

RESULTS:

The results showed that IgG antibody average value against cytomegalovirus in the blood of multiple sclerosis patients not only decreased but also was significant statistically ( $p<$ 0.05). IgM and IgG antibodies average value in the blood of multiple sclerosis patients against helicobacter pylori shown a statistically significant decrease ( $p<$ 0.05).

CONCLUSION:

Therefore it may be considered that probably helicobacter pylori presence in the individuals especially in female can alleviate MS signs. CMV infection can intensify the symptoms in multiple sclerosis patients.

Keywords

Multiple sclerosis helicobacter pylori cytomegalovirus ELISA Hamadan

1. Introduction

Multiple sclerosis (MS) is an autoimmune disease that involves the central nervous system (CNS) and its etiology is not completely explored. This disorder is frequently observed in the young aged 20 to 45 [1, 2]. High prevalence of this disease is seen in the European countries and its prevalence is average in Iran. MS disease has four distinct features include relapsing- remitting, Primary progressive, secondary progressive and progressive- recurrent [3, 4]. In spite of using many therapeutic methods for this disease as interferon prescription for alleviation of disease progression speed, faster Subsiding of attacks and reduction the annoying symptoms of MS but it is not a certain cure for this disease [5]. Genetic and environmental factors are two of the most important factors in etiology of this disease. Genetic factors include HLA genes and T lymphocytes receptor and environmental factors include bacterial, parasitic, viral and fungal infections. Pathogens proliferate in neural cells and oligodendrocytes that results in immune response and therefore it can have a role in MS. So each of the bacterial, viral, parasitic and fungal factors can be a good candidate for investigation of its role in this disease etiology [6, 7]. Many of the CNS demyelinating disorders in animal and human are related to RNA or DNA viruses. These viruses are usually enveloped viruses which acquire their envelope from the host membrane. The viruses continuously lyse CNS cells. Releasing the viruses can result in new antigens appearance from cells which are infected by viruses or cause host membrane fusion with the virus envelope and this event can result in immune response and damage the CNS. However, about MS pathogenesis, we cannot say certainly that a particular virus is related to MS and many evidences are achieved by epidemiological studies which investigate the relations between viral infection and MS. However probably most important cause in MS are viruses, High amounts of IgG gained from the brain and cerebrospinal fluid are oligoclonal bands and infection is the most important chronic inflammatory disease of CNS system [8]. CMV may has an important role in MS pathology The evidence shows that there is some relations between this virus and MS through different mechanisms as molecular mimics, and epitopic development [9]. Previous studies by researchers have shown that women who infected by helicobacter pylori compared with other women are in less danger of MS disease. HP role in different disorders in CNS system and cerebrovascular diseases is reported repeatedly This diseases includes light cognitive disorders as Alzheimer, Parkinson, Seizure disorders and migraine and probably in MS and the peripheral nervous system as polyneuropathy [10].

Regarding the daily increase of MS in Iran, etiology of this disease is very important. As it is observed pregnant mothers catch to CMV causes increasing possibility of MS in the children. The helicobacter pylori is also reported that cases with HP infection positivity and high amounts of this bacteria, do not catch this disease. So the antigens of the bacteria could be a therapeutic choice to cure MS patients

Regarding the described causes, we decided to investigate the IgM and IgG antibody average value in the blood of multiple sclerosis patients against helicobacter pylori and CMV in compare to the control group.

2. Material and methods

This study is a crosssectional study that carried out in Besat hospital of Hamadan during spring and summer 2016. The sample size calculation, was estimated by the given size formula:

$n=\frac{(Z_{1-\textstyle{\alpha\over 2}}+Z_{1-\beta})^{2}(\sigma_{1}^{2}+% \sigma_{2}^{2})}{(\mu_{1}-\mu_{2})^{2}}$

The sample size in each group was determined 45 individuals. Therefore 45 multiple sclerosis patients were choosen and 45 samples in the control group that were matched with the patient group by age and sex. After selection of the patient and controls, giving explanations to them and obtaining the written contest of patients, they were introduced to laboratory and in the suitable time and fasting situation five cc of venous blood were drawn. Then Serum samples were isolated and frozen at $-$ 70 ${}^{\circ}$ C. The earlier mentioned anti-virus antibodies and anti-bacterial antibodies were quantified by Elisa kit. The experiment was performed according to the IgM and IgG ELISA sandwich protocol (R & D Company, Germany).

2.1 Anti-cytomegalovirus IgM ELISA

In this kit, anti-human IgM antibodies were coated onto wells and during the experiment diluted samples were added to wells. In the presence of anti-cytomegalovirus IgM antibody, these antibodies would attach to wall coated antibodies. Then HRP conjugated cytomegalovirus antigen was added to detect anticytomegalovirus IgM antibody and attach to them. After washing, wells were colored in blue and the color intensity was correlated with the degree of the immunological complex in wells. The stop reagent was added and the resulting color change to yellow was quantified by Absorbance at 450 nm.

2.2 Anti-cytomegalovirus IgG ELISA

The experiment was carried out following the identical protocol as described above with the exception that coating was carried out by adding IgG antibodies and the detection was performed for IgG antibodies.

2.3 Anti-helicobacter IgG ELISA

In this kit, helicobacter antigens were coated onto wells and then during the experiment diluted samples were added to wells. In the presence of anti- helicobacter pylori antibodies, these antibodies would attach to wall coated antigens. Then HRP conjugated antiIgG antibody was added to detect herpes simplex IgG antibody and attach to them and the experiment was performed following the identical protocol as described above.

Finally statistical analyses were performed with SPSS18 (SPSS Inc, Chicago, IL, USA) and the data were analyzed using analysis of variance ANOVA and analysis of covariance ANCOVA. P values less than 0.05 are considered as significant

3. Results

In this study, 45 Relapsing-remitting multiple sclerosis patients were included based on McDonald criteria which were diagnosed by the neurologist The average age at disease onset was is 27 years and the average disease duration is 6.1 Average EDSS was between 4.5 and 2.5. There was no history of multiple sclerosis in their families.

30 of 45 multiple sclerosis patients were women (66.6%) and 15 were men (334%) and 28 of 45 control group were women (62.3%) and 17 were men (37.7%). Two groups were matched by Gender distribution based on relativity test (Table 2 and Fig. 1)

Table 1
Gender distribution in control and patients groups

Study groups	Number of	Number of
	women (%)	men (%)
Multiple sclerosis patients	30 (66.6%)	15 (33.4%)
Healthy control	28 (62.2%)	17 (37.8%)
Total	58	32

Figure 1.

Gender distribution in patients and control.

In this study, the average age was 33.3 $\pm$ 5.4 years (between the ages of 2042 years) for patients and 34.2 $\pm$ 4.8 years for control (between the ages of 2048 years). Based on the t-test which was performed for matching the age in case and control group, there was not a significant difference between control and case groups. (Table 3 and Fig. 2).

Table 2

Age distribution in patients and control

Study group	Average	Standard	Numbers
		deviation
Multiple sclerosis patients	33.3	5.4	45
Healthy control	34.2	4.8	45

Figure 2.

Age distribution in patients and control groups.

The results (of Table 4) based on SPSS analysis showed that anti-cytomegalovirus IgG antibody average value in the blood of multiple sclerosis patients with the total amount of 128 ng/ml comparing with antibody average value in the blood of control individuals with the total amount of 86 ng/ml was increased significantly ( $p<$ 0.05).

Table 3

Significantly increasing of anti-CMV IgG in multiple sclerosis patients

CMV IgG	Ab	Ab	Healthy	MS	P-value
	Average Titer	Average Titer	control no.	patients no.
	MS ng/ml	Control ng/ml
Women	122	84	28	30	0.04
Men	119	79	17	15	0.01
Total	128	86	45	45	0.02

The results (of Table 5) showed that anti-cytomegal-ovirus IgM antibody average value in the blood of multiple sclerosis patients (in women and men) with the total amount of 115 ng/ml comparing with antibody average value in the blood of control individuals with the total amount of 109 ng/ml only increased but it was not statistically significant ( $p<$ 0.05).

Table 4

Significant increasing amounts of anti-CMV IgM

CMV IgG	Ab Average Titer	Ab Average Titer	Healthy	MS patients no.	P-value
	MS ng/ml	Control ng/ml	control no.
Women	112	107	28	0.32
Men	102	96	17	15	0.35
Total	115	109	45	45	0.50

Table 5

Significantly decrease of anti- H.Pylori IgG in MS patients

H. Pylori IgM	Ab Average	Ab Average Titer	Healthy	MS	P-value
	Titer MS ng/ml	Control ng/ml	control no.	patients no.
Women	49	98	28	30	0.008
Men	62	104	17	15	0.009
Total	54	109	45	45	0.001

The results (of Table 6) showed that anti-helicobacter IgM antibody average value in the blood of multiple sclerosis patients (in women and men) with the total amount of 54 ng/ml comparing with antibody average value in the blood of control individuals with the total amount of 109 ng/ml not only increased but also it was not statistically significant ( $p<$ 0.05). Based on the Table 6 P-values of anti-helicobacter IgM antibody respectively for women, men and overall were 0.008, 0.009 and 0.001(Table 3).

The results (of Table 5) showed that anti-helicobacter IgG antibody average value in the blood of multiple sclerosis patients (in women and men) with the total amount of 62 ng/ml comparing with antibody average value in the blood of control individuals with the total amount of 112 ng/ml not only decreased but also it was statistically significant ( $p<$ 0.05).

Table 6

Significantly decrease of anti- H Pylori IgG in MS patients

H Pylori IgG	Ab Average Titer MS ng/ml	Ab Average Titer Control ng/ml	Healthy control no.	MS patients no.	P-value
Women	68	115	28	30	0.008
Men	78	109	17	15	0.001
Total	62	112	45	45	0.002

4. Discussion

The results of this study shown that anti-cytomegal-ovirus IgG antibody average value in the blood of multiple sclerosis patients not only increased but also it was statistically significant ( $p<$ 0.05). The anti-cytomegalovirus IgM antibody average value in the blood of multiple sclerosis patients only increased but it was not statistically significant ( $p<$ 0.05). The average of IgM and IgG serum antibody concentration was significantly decreased ( $p<$ 0.05). Therefore, as a result of this study probably helicobacter pylori presence in individuals especially women causes multiple sclerosis symptoms reduction. CMV infection can intensify the symptoms in multiple sclerosis patients.

In the Sanadgol et al. study at 2011, they investigated the relation between cytomegalovirus and the immunoglobulin pattern in multiple sclerosis Iranian patients with high diversity in the population (southeast of Iran) and they resulted that MS patients have higher rate of anti-CMV antibodies rate (98%) compared with the control group (52%) that it was statistically significant ( $p<$ 0.001) [11]. In the Sundqvist et al. study at 2015 which the Cytomegalovirus rate was evaluated in the multiple sclerosis patients. An epidemiological study in 658 MS patients and 786 control cases based on McDonald criteria was carried out. Anti-CMV antibody rate was quantified by Elisa kit and HLA-A and DRB1 were genotyped by SSP-PCR. The results showed that CMV is related negatively to the pathology of adult-onset of MS [12]. Our results also showed CMV role in increasing the autoimmune symptoms in MS patients. Increasing of IgG and IgM antibodies titer against CMV in the serum of the MS patients was statistically significant ( $p<$ 0.05). In the review study of Halenius and Hengel at 2014 with the title of “Human Cytomegalovirus and Autoimmune Disease”, it was pointed out that Human cytomegalovirus (HCMV) is an important member of $\beta$ -subgroup herpesvirus family and has a wide range of features as lytic replication in different tissues, continuous lifelong through latency periods and the intermitting reactivation, Extra-large proteome and immunity system manipulation (adaptive and innate). Regarding the mentioned features CMV is an important candidate for interference in autoimmune disorders [13]. These results are in the same direction with our results. Increasing of IgG and IgM antibody titer in the serum of the MS patients was statistically significant that means this virus affects autoimmune disease.

In the Pedrini et al. study at 2014, they showed that Helicobacter pylori infection in MS women is less than the healthy control group. In this study 550 MS patients and 299 selected community-based cases in the control group were included. Numbers of patients in this study are the highest numbers reported in the evaluation of the relation between H. Pylori infection and MS [14]. In our study the results also showed decreasing of IgG and IgM antibody titer against Helicobacter pylori in the serum of the MS patients which was statistically significant

In the Mohebi et al. study at 2013, they reported that patients with Helicobacter pylori infection indicate less MS outcomes which can result in this theory that H. Pylori infection probably has immunological effects or protective role in MS pathology however, more studies are needed. In this study, a strong significant inverse association between Helicobacter pylori positivity and the mean EDSS was reported These results showed that H. Pylori infection is a protective agent in Iranian CMS patients. A similar finding was observed in Li et al. study which showed that H. Pylori infection can access to a protective state in MS patients even in developed form [15]. In our study also significant decreasing of IgG and IgM antibody titer against Helicobacter pylori in the MS patients were observed

In the Gavalas et al. study at 2015, they investigated the association between Helicobacter pylori infection and multiple sclerosis and the prevalence of histologically Hp infection in 44 MS individuals were approved Concomitant diseases of autoimmune origin as hypothyroid and ulcerative colitis were particularly present in MS cases [16].

At last we can say that facing with viral infection and different bacteria as cytomegalovirus and helicobacter pylori bacteria and recurrence of it in neural cells has role in the demyelination and Inflammation and are the primers of the inflammatory neural diseases as MS and intensify it. However relation of these factors and MS etiology is proved but we cannot certainly characterize them as MS pathogenesis. It seems that for detecting the viral factors role in MS pathogenesis and finding the underlying mechanisms of these factors influence on neural systems and inflammation, more studies are needed. Infection factors determination which underlies the disease can be useful as preventive solution and suitable treatment strategy. More studies are needed during the longer period of time (one year) and bigger sample size (more patients) to achieve more accurate results and with more possibility, these two infection factors influence on multiple sclerosis must be investigated.

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