Immunoglobulin: A Natural Way to Suppress Helicobacter pylori in Humans

Adham M. Abdou, Manal M. E. Ahmed, Yusuke Yamashita, Mujo Kim

Department of Food Control, Benha University, Moshtohor, Egypt.
Department of Research & Development, Pharma Foods International Co., Ltd., Kyoto, Japan.
Department of Research & Development, Pharma Foods International Co., Ltd., Middle East Office, Cairo, Egypt.

DOI: 10.4236/health.2014.68100                

Abstract

The use of immunoglobulin is successfully applied in different areas of research, diagnostics, medical application and biotechnology. Egg yolk immunoglobulin (IgY) can successfully compete with immunoglobulin (IgG) produced in the blood of mammals. Recently, successful progresses have been achieved in Japan through industrialization of IgY technology. Using IgY has been shown to provide a safer, more efficient and less expensive method for managing disease-causing pathogens. Helicobacter pylori (H. pylori), a spiral Gram-negative microaerophilic pathogen, it infects over 50% of the population worldwide, and is recognized as the etiologic agent of gastritis, peptic ulcer, and has been linked to the development of gastric adenocarcinoma and mucosa associated lymphoid tissue lymphoma. It is found that urease is the most abundant protein of H. pylori. Urease is recognized as an essential factor in the organism colonization of the gastric mucosa. The eradication of H. pylori by administration of oral antimicrobials is not always successful and may be associated with adverse effects. Therefore, several treatment regimens have emerged to cure H. pylori infection. Accordingly, a novel approach in prevention and reduction of H. pylori infection has been reported based on production of urease-specific immunoglobulin that can suppress the bacterial colonization through urease-binding by anti-H. pylori urease IgY (IgY-urease). The use of IgY against a pathogenic factor of H. pylori will be a prudent way to suppress the infection.

Keywords

Immunoglobulin; Egg Yolk Immunoglobulin (IgY); Helicobacter pylori; Urease; Food Ingredient

1. Introduction

Helicobacter pylori (H. pylori) is a Gram-negative, microaerophilic bacterium found in the stomach. It was identified in 1982 by Australian scientists Barry Marshall and Robin Warren, who found that it was present in patients with chronic gastritis and gastric ulcers, conditions that were not previously believed to have a microbial cause. It is also linked to the development of duodenal ulcers and stomach cancer. However, over 80 percent of individuals infected with the bacterium are asymptomatic and it has been postulated that it may play an important role in the natural stomach ecology [1] .

Up to 85% of people infected with H. pylori never experience symptoms or complications [2] . Acute infection may appear as an acute gastritis with abdominal pain (stomach ache) or nausea [3] . Where this develops into chronic gastritis, the symptoms, if present, are often those of non-ulcer dyspepsia: stomach pains, nausea, bloating, belching, and sometimes vomiting or black stool [4] .

Individuals infected with H. pylori have a 10% to 20% lifetime risk of developing peptic ulcers and a 1% to 2% risk of acquiring stomach cancer [5] . Inflammation of the pyloric antrum is more likely to lead to duodenal ulcers, while inflammation of the corpus (body of the stomach) is more likely to lead to gastric ulcers and gastric carcinoma [6] .

Many questions, however, still remain concerning the optimal diagnostic and therapeutic regimens with which to approach the organism. The eradication of H. pylori by administration of oral antimicrobials, especially proton pump inhibitor based triple therapy, has been the mainstay of therapy in the past two decades. Rising antibiotic resistance increases the need to search for new therapeutic strategies; this might include prevention in form of vaccination [7] . Several non-antibiotic approaches, including probiotics, phages and phytomedicines, had been shown to be effective in treating or preventing some infectious diseases [8] . Egg yolk was an inexpensive antibody source, and the therapeutic usefulness of egg yolk immunoglobulin (IgY) in oral passive immunization had been investigated recently [9] . Immunodominant H. pylori proteins with reactivity to H. pylori specific IgY were identified in 2003 [10] . In 2004, it has been reported the successful production of anti-H. pylori urease-specific IgY [11] . Oral administration of urease-specific IgY inhibited H. pylori disease activity in H. pylori-infected gerbils, and prevented its colonization in those not yet infected [12] . Seventeen asymptomatic volunteers diagnosed as H. pylori-positive by the C¹³-urea breath test were orally administered urease-specific IgY for 4 weeks and then the breath test values were significantly decreased [13] . In a 53-year-old female administered urease-specific IgY, when combined with antacids, ameliorated gastric inflammation [14] . This article will focus on introducing the novel approaches on developing an effective, safe, and natural IgY against the H. pylori that may enable the future eradication of the organism as a significant human pathogen.

2. Epidemiology

Numerous studies have tried to assess the incidence and prevalence of H. pylori infection, its mode of transmission, and any risk factors contributing to development of the infection. The annual incidence reported in 3 adult studies in developed countries was between 0.3% and 0.5% per year [15] -[17] . Prevalence estimates vary greatly, depending on the location of the study group and the characteristics of the population studied. In general, prevalence increases with age [18] and correlates positively with a low socioeconomic status during childhood [19] . Worldwide, but especially in developed nations, infection with H. pylori is declining [20] . The acquisition of H. pylori occurs during childhood, most often by a fecal-oral or oral-oral route.

Some studies have also indicated a role for a gastro-oral route of transmission [21] . The role played by other factors, including ABO blood type, alcohol and tobacco use, dietary and nutritional influences, and genetic predisposition to infection, has also been studied, but results have been inconsistent [22] . Interestingly, a recent study of 655 subjects from a teaching hospital in Rome found an overall prevalence of infection of 40%, with a higher prevalence among nurses and auxiliary employees than among physicians [23] .

3. Pathogenicity and Virulence Factors

The earliest descriptions of the organism classified it as predominately extracellular, Gram-negative, flagellated, and motile (Figure 1). With the advancement of biochemical techniques, new information about the pathogenicity and virulence factors of H. pylori has emerged, indicating that infection by H. pylori requires a complex interaction of both bacterial and host factors.

Figure 1. Helicobacter pylori.               

Investigators have identified several bacterial proteins necessary for colonization of the gastric mucosa by H. pylori, including proteins active in the transport of the organism to the surface of the mucosa (e.g., flagellin, which is encoded on genes flaA and flaB) [24] .

Once in the presence of the gastric mucosa, bacteria induce a transient hypochlorhydria by an unknown mechanism. The urease enzyme produced by the bacteria alters the microenvironment of the organism to facilitate colonization [25] . Adherence then occurs via interaction between cell-surface glycolipids and adhesins specific to H. pylori [26] .

There also appears to be a role played by proteins called cecropins, which are produced by H. pylori and inhibit the growth of competing organisms [27] , as well as by a P-type adenosine triphosphatase, which helps prevention excessive alkalinization of the microenvironment by urease [28] . Once attached to gastric mucosa, H. pylori causes tissue injury by a complex cascade of events that depends on both the organism and the host. Like all Gram negative bacteria, H. pylori has in its cell wall lipopolysaccharide, which acts to disrupt mucosal integrity [29] . Furthermore, the organism releases several pathogenic proteins that induce cell injury. For example, the CagA protein, produced by cytotoxic-associated gene A (CagA), is a highly immunogenic protein that may be associated with more severe clinical syndromes, such as duodenal ulcer and gastric adenocarcinoma (although this question is far from settled) [30] [31] . There is increasing evidence that CagA positivity is associated with an increased risk for distal, but not proximal, gastric adenocarcinoma [32] . In addition, protein products of the vacuolating cytotoxin A gene (VacA) and the A gene induced by contact with epithelium (IceA) are known to be associated with mucosal injury [33] [34] .

Once colonization of the gastric mucosa has taken place, the immunogenic properties of H. pylori induce an inflammatory reaction with neutrophilic gastritis that ultimately results in the clinical manifestations of the infection (Figure 2). This process is mediated by host factors, including interleukins 1, 2, 6, 8, and 12; interferon gamma; tumor necrosis factor-a; T and B lymphocytes; and phagocytic cells. These factors mediate injury through release of reactive oxygen species and inflammatory cytokines [35] . Also, H. pylori appears to increase the rate of mucosal programmed cell death (also known as apoptosis) [36] .

4. General Treatment Principles

Determining the optimum treatment of H. pylori infection is difficult, because the organism lives in an environment not easily accessible to many medications and because emerging bacterial resistance presents an added challenge. Moreover, many of the recommended regimens are difficult for patients to take, leading to problems with compliance; specifically, having to take a large number of pills at least twice daily and coping with unpleasant adverse effects do little to encourage patient cooperation. Despite these obstacles, current regimens can obtain cure rates in excess of 85% in most patient populations [37] .

Antibiotic based therapy, especially proton pump inhibitor based triple therapy, has been the mainstay of therapy in the past two decades [2] . Antibiotic resistance is a well-known issue and metronidazole resistance as high as 60% - 70% was reported in some areas [38] [39] . Resistance towards clarithromycin, which initially was very effective, was also an increasing problem when its use became more popular in past decade. Even with the latest levofloxacin-based regimens, the mean eradication rate is only 80% for the resistant cases [7] . Trying to prevent the emergence of antibiotic resistance, a paradigm shift in the treatment of infectious disease had been advocated. Several non-antibiotic approaches, including probiotics, phages and phytomedicines, had been shown to be effective in treating or preventing some infectious diseases [8] . Alternative approaches in managing H. pylori infection are also necessary.

Figure 2. H & E stained section of an inflamed (infected with H. pylori) gastric antral biopsy.           

5. Novel Trend in Eradication of H. pylori Using Immunoglobulin

5.1. Egg Yolk Immunoglobulin

Immunoglobulin from yolk (IgY) is the major antibody found in hen eggs. In 1893, Klemperer first described the acquisition of passive immunity in birds, by demonstrating the transfer of immunity against tetanus toxin from the hen to the chick [40] . Three immunoglobulin classes analogues to the mammalian immunoglobulin classes; IgA, IgM, and IgG, have been shown to exist in chicken. In the egg, IgA and IgM are present in the egg white, while IgG is present in the egg yolk [41] . Immunoglobulin G (IgG) in egg yolk has been referred to as IgY to distinguish it from its mammalian counterpart [42] .

The concentration of IgY in the yolk is essentially constant (10 - 20 mg/mL) through the oocyte maturation. Approximately 100 - 400 mg IgY is packed in an egg. The concentration of IgY in the yolk is 1.23 times to the serum concentration [43] . A delay of 3 to 4 days is observed for the appearance of specific IgY in yolk after first appearance of specific IgG in the serum of a hen.

5.2. Structure and Characteristics of Avian IgY versus Mammalian IgG

General structure of IgY molecule is the same as mammalian IgG with 2 heavy (Hv) chains with a molecular mass of 67 - 70 kDa each and two light (L) chains with the molecular mass of 25 kDa each (Figure 3). The major difference is the number of constant regions (C) in H chains: IgG has 3 C regions (Cy1-Cy3), while IgY has 4 C regions (Cv1-Cv4). Due to occurrence of one additional C region with two corresponding carbohydrate chains, molecular mass of IgY (180 kDa) is larger than mammalian IgG (150 kDa). IgY is less flexible than mammalian IgG due to the absence of the hinge between Cy1 and Cy2. There are some regions in IgY (near the boundaries of Cv1-Cv2 and Cv2-Cv3) containing proline and glycine residues enabling only limited flexibility. IgY has isoelectric point 5.7 - 7.6 and is more hydrophobic than IgG [42] [44] .

Most biological effectors’ functions of immunoglobulin are activated by the Fc region, where the major structural difference between IgG and IgY is located. Therefore, Fc-dependent functions of IgY are essentially different from those of mammalian IgG. First, IgY does not activate the mammalian complement system [45] , second, IgY does not bind to protein-A and G [46] , third, IgY is not recognized by mammalian antibodies [47] i.e. rheumatoid factors (RF, an autoantibody reacting with the Fc portion of IgG) or HAMA (human anti-murine antibodies), and fourth, it does not bind to cell surface Fc receptor [48] . These differences in molecular interactions bring great advantages to the application of IgY antibodies. Then that were IgY has been successfully applied into a variety of methods in different areas of research, diagnostics, and medical areas. For these applications, IgY can successfully compete with antibodies (IgG) isolated from the blood of mammals [49] .

The advantage of usage of the immunized hen is that it produces a large number of eggs. Approximately 40 g of IgY could be collected from egg of one hen each year, compared with about 1.3 g from blood of one rabbit [50] . An industrial scale production of IgY is possible because of the availability of large number of chicken farms and automation of egg breaking and processing.

Figure 3. Structure of IgY and IgG.

Conflicts of Interest

The authors declare no conflicts of interest.

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