Helicobacter pylori – Not Only a Gastric Pathogene?

Petr Lukes1, Jaromir Astl1, Emil Pavlik2, Bela Potuznikova2, Jan Plzak1,3, Martin Chovanec1,3 and Jan Betka1 1Charles University in Prague, 1st Faculty of Medicine, Department of Otorhinolaryngology and Head and Neck Surgery, Faculty Hospital Motol, Prague 2Charles University in Prague, 1st Faculty of Medicine, Institute of Immunology and Microbiology, Prague 3Charles University in Prague, 1st Faculty of Medicine, Institute of Anatomy, Prague Czech Republic


Introduction
Helicobacter pylori is a spiral, microaerophillic, Gram-negative bacterium. Infection by H. pylori has been established as the major cause of chronic gastritis and plays important role in the pathogenesis of other gastroduodenal diseases such as peptic ulceration, gastric lymphoma, and gastric cancer (Israel and Peek 2001). H. pylori is considered to be the most common chronic bacterial infection in humans (Cave 1996). The prevalence has been estimated to range from 40 to 80% and it varies widely by geographic area, age, race, ethnicity, and socioeconomic status (Bures et al. 2006). In most cases the infection is silent, clinical manifestation appears in only 10-15% of infected individuals. This is due to different strength of virulence of H. pylori strains and different host immune system response (Stromberg et al. 2003). The stomach was supposed to be the only reservoir of infection in humans. Nevertheless H. pylori infection was detected in other sites recently. It was found in dental plaque and saliva (Kim et al. 2000) and also in oropharyngeal lymphatic tissue (Pavlik et al. 2007). This finding is of great importance because of known carcinogenic potential of H. pylori. It was declared type I carcinogen by IARC (1994). The question of direct contribution of H. pylori to oral and oropharyngeal diseases was not resolved yet.
buffer the gastric acid. H. pylori moves across gastric mucus and can adhere to epithelial cells using a variety of adhesin-like proteins (Sachs et al. 2003). Once adhered to epithelial cells, H. pylori induces a strong immune system response (Crabtree 1996). This response does not lead to elimination of the bacterium, but causes development of chronic inflammation. H. pylori is not eradicated unless an infected individual is treated with a combination of antibiotics (Portal-Celhay and Perez-Perez 2006). Chemical products of H. pylori attract cells of the immune system into lamina propria (Blanchard et al. 2004). It was shown that H. pylori can induce the maturation and activation of monocyte-derived dendritic cells. This activity is mediated by TLRs (Toll-like receptors) expressed on antigen presenting cells and leads to promotion of NK and Th1 effector responses (Portal-Celhay and Perez-Perez 2006). IFN -gamma producing Th1 polarized T cells and activated NK cells have been suggested to play an important role for development of severe pathologies (Hafsi et al. 2004). H. pylori infection in gastric mucosa is associated with the production of both proinflammatory and immunomodulatory cytokines. Changes in secretion of IL-8, IL-1beta, IL-6, TNF-alpha, TGF-beta were described (Stromberg et al. 2003). These cytokines are produced by both the immune system and epithelial cells. The response of host cells is dependent on production of H. pylori virulence factors (Blanchard et al. 2004). The most important virulence factors, which are associated with gastric diseases, are CagA (cytotoxin associated gene A) and VacA (vacuolizating cytotoxin A).

H. pylori virulence factors
Genome sequence analysis led to identification of genes encoding these virulence factors grouped in the so-called pathogenicity island (cagPAI). It is a genomic region containing about 30 genes including genes for type IV secretion system (Mobley 1996). H. pylori strains producing CagA are associated with increased risk of severe gastric pathologies compared with CagA negative strains (Portal-Celhay and Perez-Perez 2006). Injection of bacterial proteins into the gastric cells by a type IV bacterial secretion system (a multi-molecular complex that mediates the translocation of bacterial factors into the host cell) has been described (Segal et al. 1999;Oliveira et al. 2006). In this way, CagA protein can get inside the host cells and stimulate cell signalling through interaction with several host proteins. This interaction leads to increased cytokine and regulatory molecule production (Guillemin et al. 2002) and could be related to initiation of tumour transformation (Segal 1997;Tummala et al. 2004;Hatakeyama 2006). VacA is another important H. pylori virulence factor. This bacterial toxin with multiple activities is inserted into the host cell membrane, inducing cytoplasmic vacuolation (Cover and Blaser 1992). This toxin is coded by vacA gene, which is present in all H. pylori strains. Only about 50% of strains produce VacA protein. This is due to variability of vacA sequence. (Portal-Celhay and Perez-Perez 2006). There are several types of signal region (s1a, s1b, s1c, s2) and two types of midregion (m1 or m2). H. pylori strains with different forms of vacA differ in association with diseases. Strains with s1 signal sequence allele produce intact VacA toxin, s2 strains have low cytotoxic activity. Strains with s1/m1 allele combination have highest cytotoxic activity and they are associated with gastric ulceration and gastric carcinoma (Miehlke et al. 2000). s1/m2 strains are characterized by medium or low VacA production and s2/m2 strains do not produce VacA at all (Van Doorn et al. 1999). s2/m1 strains was found only sporadically (Letley et al. 1999;Martinez-Gomis et al. 2006).

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Other virulence factors are e.g. adhesins, which help H. pylori to adhere to mucosal epithelial cells (Gisbert and Pajares 2004). Important is BabA protein which binds Lewis b antigen, which is present in individuals with 0 blood group. Presence of BabA gene is connected to increased prevalence of gastric ulcers and gastric carcinoma in Lewis b positive individuals (Blanchard et al. 2004). BabA often coexists with vacA s1 and cagA alleles (Kusters et al. 2006).

H. pylori induced carcinogenesis
H. pylori i s a d e c l a r e d t y p e I c a r c i n o g e n ( I ARC, 1994). However, the exact way of carcinogenesis is not yet fully understood. There are three supposed ways of H. pylori carcinogenic action: 1. H. pylori could act as direct mutagen. Interaction of intracellular signalling molecules and H. pylori CagA may predispose cells to accumulate multiple genetic and epigenetic changes that promote multistep carcinogenesis (Hatakeyama 2006). 2. H. pylori produced VacA can cause immunosuppression by blocking proliferation of T cells (Boncristiano et al. 2003), 3. H. pylori can induce cell proliferation by increasing levels of several cytokines and regulatory molecules, which are involved in tumour formation and cell transformation (Konturek et al. 1997;Sakaguchi et al. 1999;Keates et al. 2001;Gobert et al. 2002;Schiemann et al. 2002;Wang et al. 2002). Current information about regulation mechanism of epithelial tissue by cytokines and regulatory molecules focus an interest mainly on Epithelial Growth Factor (EGF), Transforming Growth Factor (TGF) and NO synthases (NOS) (Gallo et al. 1998;Rubin Grandis et al. 1998;Sakaguchi et al. 1999;Gobert et al. 2002;Schiemann et al. 2002).

Methods of H. pylori detection in the oral cavity and pharynx
Diagnostics of H. pylori is significantly developed in gastroenterology. Attempts of H. pylori detection in other sites encountered diverse success rates (Dowsett and Kowolik 2003). Routinely used tests can be divided into non-invasive and invasive group. When detecting extragastric presence of H. pylori, invasive tests must be used based on the detection of bacteria in biopsy specimen. These invasive tests are often used to detect extragastric H. pylori presence: Histology -Several staining methods are in use. These include e.g. haematoxylin and eosin, modified Giemsa, Warthin Starry, Gimenez, and Genta (Rotimi et al., 2000). These staining methods achieve high sensitivity and specificity rates (up to 96%) (Hep 2003) in case of gastric mucosa specimens, where no other bacterial strains are supposed to be present, but provide low specificity in the case of oral specimens, where other bacterial strains are often found (Dowsett and Kowolik, 2003). Differentiation of H. pylori from other bacteria can be very difficult. Rapid Urease Test (RUT) or Campylobacter-like Organism (CLO) test is based on detection of urease production by H. pylori. When viable H. pylori bacteria are present, urea is being cleaved and the change of pH is visualized by colour indicator (Qureshi et al. 1992). This is very useful method when dealing with gastric mucosa specimens, in case of other specimens results may show high false-positive rate because of some other ureaseproducing species presence, e.g. Streptococcus spp., Haemophilus spp. a Actinomyces spp. (Dowsett and Kowolik 2003).

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Culture is currently accepted gold standard for the diagnosis of gastric H. pylori (Makristathis et al., 2004). This method achieve 80-90% sensitivity and 90-100% specificity rates (Hep 2003). Culture of H. pylori from the oral cavity or oropharynx showed to by highly difficult to perform and has met with limited success (Dowsett and Kowolik, 2003). Use of special transport medium, microaerophilic environment, supplemented media for culture and three to seven days incubation is mandatory. Overgrowth by other bacterial species often appears. Direct inhibition of H. pylori by oral species in vitro has also been reported (Ishihara et al., 1997). Transformation of H. pylori into unculturable, coccoid form in the unfavourable environment was described (Shahamat et al., 2004). Immunohistochemistry -to detect extragastric H. pylori is being used only experimentally. Tissue sections are incubated with rabbit polyclonal anti-H. pylori antibodies followed with the use of streptavidin-biotin-peroxidase kit and haematoxylin and eosin counterstaining (Akbayir et al. 2005). Molecular methods -are currently generating most possibilities of detection and also typing of H. pylori strains. Various modifications of the polymerase chain reaction (PCR) are in use. These methods are used only for experimental purposes in the detection of extragastric H. pylori. In experiments on the detection of oral and pharyngeal H. pylori many variations of PCR diagnosis has been used with a detection rate ranging between 0-90% (Dowsett and Kowolik 2003). The lack of uniformity of laboratory procedures can play a role in the reported inconsistencies. The described modification exerted different primers and probes for the detection of different DNA segments of H. pylori DNA. Various primers were used (for example, urease gene, 16S ribosomal RNA genes and others). Specificity and sensitivity of different primers, however, can vary significantly (Song et al., 1999). PCR genotyping makes it possible to distinguish different H. pylori strains and their carriage of genes encoding virulence factors (Pavlik et al. 2007). The discrepancy of published PCR results shows the importance of finding suitable PCR assay. Tissue specimens collection and especially immediate immersion into proper transport medium is essential for successful test results (Pavlik et al. 2007). It has to be considered that PCR allows the detection of a low number of bacteria or nonviable bacteria, which cannot influence progress of diseases.

H. pylori in oropharyngeal lymphatic tissue
Several studies have explored the presence of H. pylori in tonsillar and adenoid tissue. The results of these studies were inconsistent with different detection rates. The discrepancies are due to different detection methods used. Some of the methods are believed to be unsuitable for detection of extragastric H. pylori (e.g. RUT or CLO test). PCR assay is now considered the most appropriate method for detection of pharyngeal H. pylori. However, differences in the primers and probes used in published studies do not allow drawing specific conclusions. Table 1. shows an overview of published papers focused on the detection of H. pylori in tonsillar and adenoid tissue. According to above mentioned data PCR assay is considered most valuable detection method for extragastric H. pylori detection.
In their study Di Bonaventura et al. (2001) used PCR for investigation of tonsillar swabs and biopsy specimens with no evidence of H. pylori presence. Cirak et al. (2003), Bulut et al. (2006) found H. pylori in tonsillar and adenoid tissue by PCR (16S rRNA gene and glmM gene respectively). They found H. pylori strains positive for cagA gene. Bitar et al. (2005) investigated adenoid tissue specimens by RUT, histology and nested PCR (ureA gene). They found positivity by RUT and histology, but no positivity by nested PCR. In their next study www.intechopen.com these authors investigated middle ear fluids and adenoid tissue specimens using culture, RUT and PCR (ureC and adhesion subunit genes). All middle ear fluids were negative. In adenoids they found positivity by RUT, but none by PCR (Bitar et al. 2006). Yilmaz et al. (2004) found H. pylori in middle ear effusions and in one adenoid tissue specimen using PCR (23S rRNA gene). Yilmaz et al. (2006) found H. pylori in 64% of adenoid and tonsillar specimens by PCR (16S rRNA gene). Kusano et al. (2007) showed H. pylori positivity in 126 (72.9%) tonsillar specimens using PCR (16S rRNA gene). They also demonstrated the presence of coccoid forms of H. pylori in tonsillar crypts using immunoelectron microscopy. Eyigor et al. (2009) found 5,5% of adenoid and tonsillar specimens positive for H. pylori by RUT, but none of them positive by PCR (glmM gene). Vilarinho et al. (2010) found 3 adenoid and tonsillar specimens positive by RUT, 2 positive by immunohistochemistry, but none positive by fluorescence in situ hybridization or PCR (vacA gene). Abdel-Monem (2011) found 16 (53.3%) adenoid and tonsillar specimens positive by RUT and 5 (16.6%) specimens positive by PCR (ureC gene). Other studies mentioned in Table 1. used different diagnostic methods with different detection rates.
The relationship between H. pylori infection and gastric tumour pathogenesis has been well described. It was supposed that H. pylori could act the same way in progression of oropharyngeal tumourigenesis. Some authors tried to identify a correlation between H. pylori and cancers of head and neck (Table 2.). Tests which determined serum levels of anti-H. pylori antibodies in patients with head and neck spinocellular carcinoma (HNSCC) brought inconsistent results (Grandis et al. 1997;Aygenc et al. 2001;Rubin et al. 2003;Nurgalieva et al. 2005). Okuda et al. (2000) proved the presence of H. pylori in oral swab specimens and oral cancer specimens using RT PCR (reverse transcriptase polymerase chain reaction) and culture. On the other hand Kanda (2005) found no HNSCC specimen positive using PCR, culture and immunohistochemical analysis. Kizilay et al. (2006) did not find H. pylori in laryngeal SCC and non-neoplastic specimens using haematoxylin and eosin stain or modified Giemsa stain. Akbayir et al. (2005) found H. pylori in specimens collected from laryngeal cancers and benign laryngeal disorders by histopathological methods, but not by immunohistochemical methods. Only one study performed PCR genotyping of H. pylori strains in specimens collected from the oropharynx. Tonsillar tissue specimens were collected from patients with chronic tonsillitis, obstructive sleep apnea syndrome (OSAS) and tonsillar cancer. The detected H. pylori strains differ from strains found in the stomachs of Czech patients with gastric diseases (Pavlik et al. 2007).

Comparison of oral and oropharyngeal genotypes
It is supposed that H. pylori is spread from person to person by oral-oral or faecal-oral route (Brown 2000), this hypothesis has not yet been convincingly demonstrated. Assuming the oral cavity and oropharynx as a gateway of infection, we can assume that in the oral cavity and oropharynx of the same individual we can find H. pylori strains of the same genotype. Initial works focused on comparison of oral and gastric H. pylori strains used endonuklease restriction analysis, single strand conformation polymorphism analysis (SSCP) or PCR (Shames et al. 1989;Khandaker et al. 1993;Zhang and Lu 1997;Kim et al. 2003). Identical strains have been found in gastric mucosa and oral cavity. The first comparison of gastric and oral H. pylori strains using PCR genotyping performed Wang et al. (2002) and, consequently, Burgers et al. (2008). Different genotypes in the stomach and oral cavity were found in both studies. PCR assays used by these authors could be considered more accurate     Burgers et al. (2008), who report that only 38% of persons with demonstrated presence of H. pylori in the oral cavity also had the infection in the stomach. These authors also reported the finding of 10 cases with positive H. pylori in saliva, with no detectable specific anti-H. pylori antibodies in serum. This is consistent with the results obtained by Lukes et. al. (2009). H. pylori was found in the oropharynx in 12 patients with no demonstrable antibody response.

Conclusions
Oral cavity (saliva and dental plaque) is now considered a possible extragastric reservoir of H. pylori. pylori infection in other locations independently to the gastric infection. This should be considered in future epidemiological studies. Not only antibodies should be evaluated but also identification of the exact location of the infection must be done.
In the future it would be appropriate to focus attention on local effects of H. pylori in oropharyngeal lymphoid tissue. Changes in the expression of some cytokines caused by H. pylori, which were described in the gastric mucosa, can be expected in the oropharyngeal tissue. Another study focused on oropharyngeal H. pylori genotyping should be done. In case that high virulent H. pylori strains can survive in oropharyngeal tissue, translocation of toxins into the oropharyngeal mucosa cells with subsequent cytokine response can be expected. Nevertheless this assumption has not been confirmed nor refuted yet.

Acknowledgements
This work was supported by grant NT11523 of the Internal Grant Agency of the Ministry of Health of the Czech Republic