!e Controversy over Anti-Helicobacter pylori !erapy

Helicobacter pylori is a Gram-negative spiral-shaped bacterium, member of ε-Proteobacteria specifically colonizing the gastric epithelium of humans. It causes one of the most common infections worldwide, a"ecting about half of the world’s population. However, it should be noted that the prevalence of H. pylori, particularly in the Western world, has significantly decreased coinciding with an increase of some autoimmune and allergic diseases, such as asthma. Various epidemiological studies have also documented a negative association between H. pylori colonization and the presence of GERD (gastroesophageal re/ux disease) and risk of esophageal cancer. Additionally, an upward trend of obesity recently observed in inhabitants of developed countries raised a question about the relationship between H. pylori infection and the human body mass index. #e first part of this review describes common, recommended anti-H. pylori treatments. #e second part, presents the results of recent experiments aimed at evaluating the association between H. pylori infections and gastro-esophageal diseases, the level of stomach hormones, the human body mass index and allergic diseases. Although some studies suggest an inverse association of H. pylori infection with some health problems of the modern world such as asthma, obesity or GERD, H. pylori should be considered as a harmful human pathogen responsible for serious and sometimes lethal diseases. #us, many scientists advocate the eradication of H. pylori. K e y w o r d s: Helicobacter pylori, allergy, GERD, NAP protein, obesity, therapy Bocian K.M. and Jagusztyn-Krynicka E.K. 4 240 promoters in gastric mucosa, which are known to be methylated in cancer patients (Cover and Blanke, 2005; Ding et al., 2010; Douraghi et al., 2008; Tegtmeyer et al., 2011; Yamaoka, 2008). Sequencing of several H. pylori genomes and comparative genomics experiments revealed a high level of genome diversity. At first, by comparing the genomes of 15 H. pylori strains Salama et al. determined that H. pylori genes constituting the core set count 1 281 genes (Salama et al., 2000). #e growing number of genomes included in microarray analyses resulted in redefining the number of core genes in H. pylori genomes. At present, it is widely assumed that H. pylori core genes consist of about 1 100 genes. Auxiliary genes amount to 22–27% of the genome, encoding mainly proteins of unknown function, the Cag protein, outermembrane proteins (OMP) and proteins involved in DNA metabolism (Dong et al., 2009; Gressmann et al., 2005). Some strain-specific genes are disease-specific. #e diversity of H. pylori genomes is not only noticeable when evaluating the number of common genes, but also when examining gene nucleotide sequences. Genetic variety among H. pylori strains arises from intra-genomic diversifications (for example – point mutations, recombination and slipped-strand mispairing) as well as inter-genomic recombination – the ability of H. pylori to take up exogenous DNA and incorporate it into its genome (Dorer et al., 2009). #e process allows the pathogen to adapt to various niches within the same host or to the changing environments during long-lasting infections. Recommended anti-Helicobacter therapies According to several international guidelines, different drug regimens are recommended for treatment of H. pylori infections. #ese include triple, quadruple, (sequential or concomitant) therapy regimens. Treatment regimen should be selected according to areas of low or high claritromycin resistance. Standard triple therapy which involves administration of a proton pump inhibitor combined with clarithromycin and amoxicillin or metronidazol for 7 to 14 days was the most commonly recommended first-line treatment by guidelines published in Europe and North America since the mid 1990s (Chey and Wong, 2007; Malfertheiner et al., 2012). However, the recent data showed that this drug combination has lost some efficacy and according to recently published worldwide guidelines should be abandoned when the clarithromycin resistance rate in the region is more than 15–20% (Malfertheiner et al., 2012). Increased doses of proton pump inhibitors had small e"ects on eradication rates (Fuccio et al., 2007; Vakil and Connor, 2005). Bismuthbased quadruple and levo/oxacin-based triple regimens are also frequently recommended, but as second-line therapies. #ird-line options (empiric regimes tailored to individual antibiotic sensitivities) include treatments based on rifabutin (an antituberculous agent) and furazolidone. However, susceptibility testing is not common, but, when it is employed, it is only carried out in specialist research-oriented centers. Sequential therapy is an alternative to standard triple therapy for eradication of H. pylori (Gisbert, 2010; Gisbert et al., 2010). It aims to overcome clarithromycin resistance. During the first stage of therapy, amoxicillin is administered to weaken the bacterial cell wall, which other wise prevents the formation of channels that block clarithromycin from entering the bacterial cell and, in e"ect, cause resistance to the antibiotic. Subsequently, clarithromycin and nitroimidazole are administered for a further 5 days in the second phase of the therapy. Administration of the proton-pump inhibitor is continuously employed throughout the treatment. Generally, the sequential therapy has a better rate of curing H. pylori infection than classical triple therapy. However, clinically the sequential administration of the two drug combinations is relatively complex. As an alternative the concomitant quadruple therapy including the same four drugs as sequential therapy given concomitantly has been tested. It appears to be equally e"ective as sequential therapy but less complex (Essa et al., 2009; Wu et al., 2010). Treatment against H. pylori still fails in more than 20% of patients and a more acceptable eradication level is greatly anticipated. Two main obstacles against e"ective therapy are the resistance of H. pylori to di"erent antibiotics (i.e. clarithromycin, metronidazole, amoxicillin, levo/oxacin), correlated with the consumption of antibiotics in the general population, and lack of strict abidance to the rules of drug administration, which involves the e"orts of both doctor and patient. #e prescribed defined therapy is long and complicated. It is uncomfortable for patients and impedes complying with the recommended procedures. In view of these facts, there is an urgent need to intensify the fight against H. pylori by developing alternative methods of treating Helicobacter pylori infections (De Francesco et al., 2010; O’Connor et al., 2010). It was documented that the addition of probiotics to a standard antibiotic treatment improved slightly H. pylori eradication rate and considerably reduced therapy-associated adverse e"ects (Lionetti et al., 2010; Vitor and Vale, 2011; Zou et al., 2009). #e alarming rise of antibiotic resistant pathogenic microorganisms renewed interest in antibacterial, including anti-Helicobacter, research and forced scientists to search for new drugs with novel modes of action. Some new anti-Helicobacter drugs are currently under Anti-Helicobacter therapy 4 241 development. New, potentially e"ective agents should fulfill several requirements, at least display strong, specific antibacterial activity against Helicobacter, when used in mono-therapy, and exhibit activity in low pH. Examples of such agents are: the TG44 molecule synthesized by the Nagase ChemeteX Corporation and acyl-lysyl oligomers (OAKs). #e former compound, tested so far only in in vitro experiments, is a highly specific anti-Helicobacter molecule, which activity is based on the disruption of the cell outer membrane (Kamoda et al., 2006). Latter compounds are synthetic antimicrobial peptides (AMPs) of broad specificity, which demonstrate high efficacy against Helicobacter in in vitro and in vivo tests (Makobongo et al., 2009; Makobongo et al., 2012). #ey are a unique and diverse group of molecules produced by many tissues and cell types of various organisms. As AMPs have recently elicited interest as new antibacterial drugs, one can expect that new AMPs will be tested as anti-Helicobacter agents soon (Brogden, 2005). However, it should be kept in mind that AMP therapy might potentially enable pathogens to overcome the innate immune response of an immunocompetent host (Brodsky and Gunn, 2005). Unexpected consequences of Helicobacter pylori infection/eradication Impact of Helicobacter pylori infection on esopha geal diseases. Symptoms of the gastro-esophageal re/ux disease (GERD) appear when the impairment of motility of the gastric system allows for the contact of the gastric content with the esophagus epithelium. #is may lead to development of Barrett esophagus (BE), which is recognized as a risk factor for the subsequent development of esophageal adenocarcinoma (EAC). #e pathogenesis of these diseases is complex and multifactorial, but acidity of the re/uxate is a crucial factor inducing GERD development. #e decreasing prevalence of H. pylori infections and related diseases, especially in developed countries, and, at the same time, an increase in the recognition of gastro-esophagal re/ux symptoms and its complications, raise a question whether H. pylori is a likely etiological factor for this changing epidemiology. #is phenomenon has been observed in many countries, at di"erent geographic locations, such as Japan or USA (Blaser, 2008; Kim et al., 2011; Rajendra, 2011; Yang et al., 2009). #e causal relationship between H. pylori infections and the gastro-esophageal disease has been examined in a large number of epidemiological studies. #e first observations were made by Labenz et al., who reported that H. pylori eradication in patients with duodenal peptic ulcer stimulates the development of re/ux eosophagitis (Labenz et al., 1997). Comparative analyses concentrated on assessing the rate of H. pylori infection in patients with re/ux esophagitis compared to those with a normal esophagus as well as on the evaluation of the e"ect of H. pylori eradication on the development of re/ux esophagitis. Although some studies confirmed the inverse associations between these two analyzed phenomena, contradictory results also have been reported. For more details we recommended review papers, such as by Graham et al., 2007; Hung and Wong, 2009; Sharma and Vakil, 2003; Souza and Lima, 2009. Lack of consensus between various studies may be due to many factors, such as difficulties in evaluating the clinical aspects of H. pylori infection or the di"erences in the genotype of the infecting strain. It was documented that H. pylori can colonize various parts of the stomach and that the site of infection is an important factor in/uencing the consequences of colonization. Antrum gastritis is related to high in/ammation and high acid secretion, whereas corpus gastritis correlates with low acid secretion. Another important factor is the genetic and immunological status of the host. #ree recent meta-analysis studies conducted by Islami and Kamangan, Qian et al. and Yaghoobi et al. summarized the data published during last twenty years (Islami and Kamangar, 2008; Qian et al., 2011; Yaghoobi et al., 2010) Islami and Kamangar used 19 carefully selected studies to examine the association between H. pylori infection and esophageal adenocarcinoma (EAC). Presented data suggested inverse association of the CagA-positive H. pylori colonization with risk of EAC. Meta-analysis of Yaghoobi et al. evaluated the risk of GERD development due to H. pylori eradication and showed that the frequency of GERD does not increase a<er H. pylori eradication among dyspeptic patients, whereas a two-fold higher risk of GERD development in patients with peptic ulcers was observed. Meta-analysis conducted by Qian et al. did not show any association between H. pylori eradication and the occurrence of symptomatic GERD. Potential mechanism of the protective e"ect of H. pylori gastric colonization against esophageal diseases still remains unexplainable. Apart from changes in gastric acidity, stomach colonization by H. pylori in/uences the level of at least two hormones: leptin and ghrelin (see below), which can in/uence the esophagus epithelium as esophagus cells contain leptin receptors. #e recent progress in sequencing technology in combination with the development of new bioinformatic tools allows us to study the microbiome (a set of bacterial genes present in a specific ecological niche) of the stomach and esophagus. It is expected that this strategy will permit tracing changes in the microbiome that are correlated with disease development or are due to H. pylori eradication. Analysis of the stomach microbiota revealed that it is much more complex than it was assessed before. H. pylori was found to be Bocian K.M. and Jagusztyn-Krynicka E.K. 4 242 the most abundant phylotype in the stomach of individuals tested as H. pylori-positive by standard methods (Bik et al., 2006). Structure of the human gastric bacterial community was determined to be dependent on the H. pylori-induced disease. For instance, the stomach microbiota of gastric cancer patients di"ers significantly compared to microbiota of dyspeptic individuals (Dicksved et al., 2009; Maldonado-Contreras et al., 2011). Metagenomic study of the esophagal micro biome also revealed its astonishing complexity and significant changes in the microbiome structure connected to pathological alterations of the esophageal epithelium (Yang et al., 2009). As even short term antibiotic treatment of H. pylori infections has tremendous repercussions for the gut microbiome structure, it may also be a factor of GERD development (Jakobsson et al., 2010). Further metagenomic studies are required to shed more light on the contribution of stomach or esophagus dysbiosis on disease development. Correlation between Helicobacter pylori infection and asthma and allergic diseases. In recent years, a rise in the prevalence of bronchial asthma in developed countries has been observed. Many environmental factors, such as tobacco smoke, air pollution or allergen exposure, are without doubt responsible for this documented upward tendency. However, the in/uence of human microbiota on allergic diseases should be also taken into account. As over the past years the prevalence of H. pylori infection has been decreasing, the causal relationship between H. pylori infection and asthma was carefully evaluated in many epidemiological studies. #e relationship between these two diseases was noted for the first time in 1997 by Kosunen et al. (Kosunen et al., 1997). Although the conducted studies provided controversial results, it is rather accepted that human colonization with H. pylori CagA-positive strains may have an inverse e"ect on development of bronchial asthma. However more studies are required to prove a real association between H. pylori eradication in childhood and subsequent development of asthma (Hung and Wong, 2009). At this point it should be also pointed out that H. pylori CagA positive strains which potentially may be protective against GERD or asthma are strongly associated with gastric cancer, which is the second leading cause of cancer-related deaths worldwide (Huang and Hunt, 2003). For more data from epidemiological studies see papers cited by Roussos et al., D’Elios et al. and Malferheiner et al. (D’Elios et al., 2009; Malfertheiner et al., 2011; Roussos et al., 2005). Despite the lack of a clear hypothesis explaining the link between these two diseases, some data suggest that this inverse association might be due to the di"erences in the type of immune response induced. According to the WHO definition, asthma is a chronic in/ammatory disease of the airways associated with a predominant activation of CD4+ #2 lymphocytes, which produce several #2 cytokines, including IL-4 and IL-5 (Del Prete et al., 1993; Robinson et al., 1992). In contrast, H. pylori gastric colonization preferentially elicits a #1 mucosal immune response with the production of IFN-γ, IL-12, IL-18, IL-23 and TNF-α (D’Elios et al., 2009). H. pylori neutrophil-activating protein (HP-NAP) is a main H. pylori virulence factor responsible for this e"ect. HP-NAP is a 200 kDa ballshaped dodecamer formed by four-helix bundled subunits (17 kDa) with a hollow central part (Tsuruta et al., 2012). Structurally it belongs to the Dps (DNA protecting protein under starved condition) protein family. #e role of this protein in bacterial cells is still controversial. Analysis of its ability to bind to DNA resulted in incoherent data. Additionally, although HP-NAP is a bacterioferritin able to bind up to 500 atoms of iron per dodecamer, the role of this process in bacterial physiology remains unclear. Furthermore, HP-NAP was described as a cytoplasmic protein, released a<er cell lysis. Once released in the gastroduodenal mucosa, NAP is transported via transcytosis across endothelial cells, (de Bernard and D’Elios, 2010) stimulating subsequently human neutro phils, monocytes and dendritic cells via activation of the Toll-like receptor 2 (TLR2). In consequence, high upregulation of both the production of IL-12 and IL-23 occurs (D’Elios and de Bernard, 2010). HP-NAP activity also causes the decrease of IL-4-secreting cells. As a result, HP-NAP supported by other H. pylori factors induces the production of IL-12 and IL-23 that both promote the preferential development of #-1 cells and repress the #-2 allergic response (Amedei et al., 2010; Amedei et al., 2006; Cappon et al., 2010). Administration of HP-NAP has a beneficial e"ect in case of asthma. Arnold et al. proved that H. pylori infections induced T regulatory cells (Tregs) and protected mice from asthma, especially when mice were infected neonatally. A<er H. pylori eradication due to antibiotic treatment, the protection e"ect was abolished (Arnold et al., 2011). #e e@cacy of HP-NAP against asthma was also confirmed by D’Elios et al., who documented that systemic and mucosal administrations of HP-NAP result in reduction of the amount of eosinophil cells, immunoglobulin E and #2 cyto kines in the mice bronchitis model. #is suggests that H. pylori infection is able to induce long lasting #1 type of immune response. Taking the above into account, HP-NAP seems to be an e"ective factor for prevention and treatment of asthma and allergic diseases (D’Elios et al., 2009). Apart from playing a role in induction of #1 in/ammation and inhibition of the #2 response, HP-NAP may potentially be used in cancer therapy. As a very Anti-Helicobacter therapy 4 243 powerful inducer of IL-12 and IL-23, HP-NAP represent the most e"ective cytokine in regard to tumor eradication, anti-metastatic activity and long-term anti-tumor immunity (Colombo and Trinchieri, 2002). IL-12 was recently ranked third in a comprehensive list of immunotherapeutic agents with high potential in treating cancer. Codolo and colleagues showed that local administration of HP-NAP decreases tumor growth by triggering tumor necrosis in a mouse model of bladder cancer. HP-NAP-treated tumors show also a reduced vascularization due to the anti-angiogenic activity of IFN-γ induced by treatment of cancer implants with HP-NAP (Codolo et al., 2012a; Codolo et al., 2012b). In sum it should be pointed out that even persistent infection with H. pylori may be linked to protection from some autoimmune diseases, it is not recommended to leave H. pylori infection untreated in asthmatic patients. (D’Elios and de Bernard, 2010). However based on the performed studies it is tempted to speculate that administration of rHP-NAP might be beneficial not only against allergic diseases, but also to fight cancer. Impact of H. pylori infection and anti-Helicobacter therapy on obesity. #e decreasing prevalence of H. pylori infections during the second half of the 20 century and the beginning of the 21 is noticeable across the whole developed world. For example, in some countries currently less than 10% of school children are carriers of this microorganism (Chen and Blaser, 2008; Rothenbacher et al., 1998; Segal et al., 2008). At the same time, the incidence of obesity among the same population group has been observed. It prompts many research groups to evaluate the impact of H. pylori infection on body weight. Two hormones, leptin and ghrelin, play a crucial role in body weight balance by regulating food intake and energy disbursement. Leptin is a 16 kDa protein, the product of the ob gene, which is synthesized and secreted mainly by adipocytes (Zhang et al., 1994). However, it has recently been shown that this hormone is also present in rat and human gastric mucosa (Bado et al., 1998; Sobhani et al., 2000). As leptin deficiency causes obesity in humans and mice and since H. pylori induced gastritis may in/uence the leptin level, many comparative epidemiological studies have recently been conducted to examine the e"ect of H. pylori infections on gastric leptin expression and on the body mass index (BMI). Most of these studies compared infected and non-infected individuals by CagA-positive and CagAnegative strains. Infection by CagA-positive H. pylori strains was shown to result in more severe gastritis and more o<en led to gastric cancer than infection by CagA-negative H. pylori. Majority of obtained data has not implied any associations between the plasma leptin level and H. pylori infection (Azuma et al., 2001; Chuang et al., 2009; Ioannou et al., 2005). However, a study examining prepubertal children conducted by Pacifico et al. indicated that the serum leptin level was significantly lower in H. pylori positive patients than in H. pylori negative individuals (Pacifico et al., 2008). In contrast to data concerning the plasma leptin level, many investigators provided convincing results showing that H. pylori infection results in a significant increase of gastric leptin expression (Azuma et al., 2001; Jun et al., 2007) what, in turn, might led to weight loss. Con/icting results have also been published regarding the e"ect of H. pylori infection on ghrelin level. Ghrelin is a 28 amino acid peptide predominantly produced by the stomach, which is thought to be the most potent growth hormone releaser (Kojima et al., 1999). Ghrelin has also been implicated in the control of food intake (Nakazato et al., 2001). It was determined to a"ect metabolic functions and evoke weight gain (Wren et al., 2001). In the study performed by Gokcel et al., no di"erences in the plasma ghrelin concentration between adults positive and negative for H. pylori have been observed (Gokcel et al., 2003). In contrast, many studies performed on Japanese patients revealed a strong negative impact of H. pylori infection on the plasma ghrelin level, which was positively correlated with gastric ghrelin mRNA expression (Isomoto et al., 2005a; Isomoto et al., 2005b; Shiotani et al., 2005). Additionally, some analyses indicated that this e"ect is dependent on the severity of the disease and in/uenced by the patient’s gender (Chuang et al., 2009). Although H. pylori infection modulates the plasma ghrelin and stomach leptin level, epidemiological studies do not document any associations between H. pylori infection and the body max index of the analyzed patients. A large US-based population study showed that there is no correlation between the presence of H. pylori and the genetic status of the colonizing strain (CagA-positive vs. CagA-negative strains) as well as the weight of the analyzed individual (Cho et al., 2005; Ioannou et al., 2005). #e second set of experiments was directed towards analyzing the e"ect of H. pylori eradication, plasma leptin or ghrelin level and the increase in BMI. #ey provided rather consistent results. In many studies, it has been reported that H. pylori eradication is associated with the increase of the circulating leptin level, the decrease of the ghrelin level resulting in an increase in BMI. #is e"ect was independent of the examined population and was observed both among adult Japanese as well as American individuals who underwent antiHelico bacter therapy. Additionally, similar long-term e"ect of H. pylori eradication was observed among prepubertal children (Francois et al., 2011; Fujiwara et al., 2002; Osawa et al., 2006; Pacifico et al., 2008). However, Bocian K.M. and Jagusztyn-Krynicka E.K. 4 244 Suto et al. noticed that the e"ect of H. pylori eradication on BMI is dependent on the serum pepsinogen I/II ratio (Suto et al., 2009). Interestingly, experiments conducted by Nwokolo et al., who for the first time examined the e"ect of H. pylori eradication in H. pylori asymptomatic patients, showed a significant increase of the ghrelin level a<er therapy. Authors have thus concluded that lowering the prevalence of H. pylori infections in developed countries might lead to the observed increase in obesity (Nwokolo et al., 2003).