Rafaela Sierra

Distribution of Open Reading Frames of Plasticity Region of Strain J99 in Helicobacter pylori Strains Isolated from Gastric Carcinoma and Gastritis Patients in Costa Rica

ABSTRACT The plasticity region of Helicobacter pylori strain J99 is a large chromosomal segment containing 33 strain-specific open reading frames (ORFs) with characteristics of a pathogenicity island. To study the diversity of the plasticity region, 22 probes corresponding to 20 ORFs inside the plasticity region and two ORFs on its boundaries were hybridized to genomic DNA isolated from clinical strains of H. pylori from patients with gastritis or gastric adenocarcinoma. Highly variable hybridization patterns were observed. The majority of the clinical strains presented a hybridization profile similar to that of J99; thus, these ORFs are not J99 strain specific. No association was found between a particular hybridization pattern and the clinical origin of the strain. Nevertheless, two single ORFs (JHP940 and JHP947) were more likely to be found in gastric cancer strains. They may be new pathogenicity markers. An in vitro expression study of these ORFs was also performed for the J99 strain, under different conditions. Thirteen ORFs were consistently expressed, six were consistently shut off, and three were expressed differentially. Most of the constitutionally expressed genes were located on the 3′ part of the plasticity region. Our results show that the plasticity region, rather than being considered a pathogenicity island per se, should be considered a genomic island, which represents a large fragment of foreign DNA integrated into the genome and not necessarily implicated in the pathogenic capacity of the strain.

The cag Pathogenicity Island of Helicobacter pylori Is Disrupted in the Majority of Patient Isolates from Different Human Populations

ABSTRACT The cag pathogenicity island (cag-PAI) is one of the major virulence determinants of Helicobacter pylori. The chromosomal integrity of this island or the lack thereof is speculated to play an important role in the progress of the gastroduodenal pathology caused by H. pylori. We determined the integrity of the cag-PAI by using specific flanking and internally anchored PCR primers to know the biogeographical distribution of strains carrying fully integral cag-PAI with proinflammatory behavior in vivo. Genotypes based on eight selected loci were studied in 335 isolates obtained from eight different geographic regions. The cag-PAI appeared to be disrupted in the majority of patient isolates throughout the world. Conservation of cag-PAI was highest in Japanese isolates (57.1%). However, only 18.6% of the Peruvian and 12% of the Indian isolates carried an intact cag-PAI. The integrity of cag-PAI in European and African strains was minimal. All 10 strains from Costa Rica had rearrangements. Overall, a majority of the strains of East Asian ancestry were found to have intact cag-PAI compared to strains of other descent. We also found that the cagE and cagT genes were less often rearranged (18%) than the cagA gene (27%). We attempted to relate cag-PAI rearrangement patterns to disease outcome. Deletion frequencies of cagA, cagE, and cagT genes were higher in benign cases than in isolates from severe ulcers and gastric cancer. Conversely, the cagA promoter and the left end of the cag-PAI were frequently rearranged or deleted in isolates linked to severe pathology. Analysis of the cag-PAI genotypes with a different biogeoclimatic history will contribute to our understanding of the pathogen-host interaction in health and disease.

Association of interleukin-1B and interleukin-1RN polymorphisms with gastric cancer in a high-risk population of Costa Rica.

Several risk factors have been associated with gastric cancer, among them Helicobacter pylori infection. This bacterium yields inflammation, the degree of which depends on the bacterial strain and the severity of the host response. The inflammatory response involves a complex cytokine network. Recently, polymorphisms of the genes coding for interleukin-1β (IL-1B), interleukin-1Ra (ILRN) and interleukin-10 have been associated with an increased risk of gastric cancer. In order to determine the association of the IL-1B, IL-1RN and IL-10 polymorphisms with gastric cancer in a high-risk Costa Rican population, we analysed purified DNA of 58 gastric cancer patients, 99 controls and 41 patients classified as group I or II, according to the Japanese classification. Genotyping was carried out by PCR, PCR-RFLP and pyrosequencing analysis. We did not find any association of the IL-1B-31, IL-1B-511 and IL-10 polymorphisms with the risk for developing gastric cancer in the studied population. Carriers of the IL-1B+3954T/– had an increased risk for developing gastric cancer (OR 3.7; 95%CI: 1.34–10.2). Also we found an increased risk for developing gastric cancer for allele 2 heterozygotes of the IL-1RN (OR 2.94; 95%CI: 1.09–7.93). This is the first time that IL-1B+3954 has been associated with gastric cancer. This is one of the first studies trying to describe the role played by IL-1B, IL-1RN and IL-10 genetic polymorphisms in gastric cancer in one of the highest risk American countries. Further investigation on American countries is needed.

Composition and Gene Expression of the cag Pathogenicity Island in Helicobacter pylori Strains Isolated from Gastric Carcinoma and Gastritis Patients in Costa Rica

ABSTRACT The composition and in vitro expression of the cagpathogenicity island genes in a group of Helicobacter pylori strains obtained from patients suffering from chronic gastritis-associated dyspepsia (n = 26) or gastric carcinoma (n = 17) were analyzed. No significant difference in the distribution of the 10 studied regions was found between the cases and the controls. Nine strains did not harbor any of the selected regions: eight (30.8%) isolated from patients with gastritis only and one (5.9%) from a patient with gastric carcinoma. No association was found between the number of repeated sequences at the 3′ end of the cagA gene or the presence of tyrosine phosphorylation motifs and the clinical origin of the strains. ThevirB10 homolog gene was the sole gene studied to be significantly expressed more often in cancer strains than in gastritis strains (P = 0.03).

Urokinase plasminogen activator receptor is expressed in invasive cells in gastric carcinomas from high- and low-risk countries

Gastric cancer is the second cancer causing death worldwide. Both incidence and mortality rates vary according to geographical regions. The receptor for urokinase plasminogen activator (uPAR) is involved in extracellular matrix degradation by mediating cell surface associated plasminogen activation, and its presence on gastric cancer cells is linked to micro‐metastasis and poor prognosis. Immunohistochemical analyses of a set of 44 gastric cancer lesions from Costa Rica showed expression of uPAR in cancer cells in both intestinal subtype (14 of 27) and diffuse subtype (10 of 17). We compared the expression pattern of uPAR in gastric cancers from a high‐risk country (Costa Rica) with a low‐risk country (Norway). We found uPAR on gastric cancer cells in 24 of 44 cases (54%) from Costa Rica and in 13 of 23 cases (56%) from Norway. uPAR was seen in macrophages and neutrophils in all cases. We also examined the nonneoplastic mucosa and found that uPAR was more frequently seen in epithelial cells located at the luminal edge of the crypts in cases with Helicobacter pylori infection than in similar epithelial cells in noninfected mucosa (p = 0.033; χ2 = 4.54). In conclusion, the expression of uPAR in cancer cells in more than half of the gastric cancer cases suggests that their uPAR‐positivity do not contribute to explain the different mortality rates between the 2 countries, however, the actual prevalence of uPAR‐positive cancer cells in the gastric cancers may still provide prognostic information.

Association of serum pepsinogen with atrophic body gastritis in Costa Rica.

Individuals with atrophic gastritis (AG), especially atrophic body gastritis (ABG), are at increased risk of developing gastric cancer. Serum concentrations of pepsinogens (PG) have been proposed as markers for ABG. The aim of this study was to determine the risk factors for AG and ABG and the potential of using serum PG concentrations to detect ABG in a dyspeptic population in Costa Rica, which is one of the countries with the highest incidence and mortality rates of gastric cancer in the world. Seven biopsy specimens, a fasting blood sample and a questionnaire concerning sociodemographic factors were obtained from 501 consecutive dyspeptic patients. The serum PGI level and the PGI/PGII ratios were significantly lower in patients with ABG than in other groups (P<0.000). A cut-off point of 3.4 led to a sensitivity of 91.2% in identifying ABG, a negative predictive value of 98.1%, but a positive predictive value of only 11.2%. Helicobacter pylori were present in 93% of the patients and all those with peptic ulcers were positive. AG was associated with increased age, lower body mass index, high alcohol intake and low fruit consumption. ABG was associated with age, alcohol consumption and PGI/PGII<3.4. In dyspeptic patients with a high prevalence of H. pylori infection, serum PG levels provide an assessment of ABG but it is necessary to introduce other serological and genetic markers in order to achieve a better specificity. Those markers could be serum antibodies to H. pylori-CagA, cytokine gene polymorphisms or others.

X-ray screening seems to reduce gastric cancer mortality by half in a community-controlled trial in Costa Rica

X-ray screening of gastric cancer is broadly used in Japan, although no controlled trial has proved its effectiveness. This study evaluates the impact of an X-ray screening demonstrative intervention to reduce gastric cancer mortality in a Costa Rican region. The evaluation follows a quasi-experimental, community-controlled design, with measures before and after. About 7000 individuals participated by invitation in the two-wave screening programme. X-ray screening was followed by videoendoscopy and gastric biopsies. Treatment included resection with or without lymph node dissection. Comparisons with two control groups estimate that gastric cancer mortality was halved in the period from 2 to 7 years after the first screening visit. Validity of X-rays as used in this intervention had 88% sensitivity, 80% specificity, and 3% predictive value for individuals with two screening visits. Incidence in the screened group increased up to four times. Case survival was 85% in the intervention group after 5 years, compared to 12% among the controls before the intervention and 35% among the controls in the same region after the intervention. Although X-ray mass screening seems able to reduce stomach cancer mortality, its high cost may be an obstacle for scaling up this intervention in a non-rich country like Costa Rica.

Helicobacter pylori in a Costa Rican dyspeptic patient population.

Gastric biopsies from 65 Costa Rican dyspeptic patients were investigated for the presence ofHelicobacter pylori DNA by polymerase chain reaction (PCR) amplification of 16S rRNA sequences. Both frozen and paraffin-embedded samples were used, and the results were compared with bacterial culture and histological examination.Helicobacter pylori DNA was detected by PCR in 60 (92 %) of the patients, andHelicobacter pylori strains confirmed by PCR could be isolated from 37 of them. Altogether, 59 patients were shown to be infected using the combination of culture and histology as the reference method. The sensitivity of PCR analysis of frozen material was 98 % (58/59). The PCR analysis of paraffin-embedded samples seemed less reliable than that of frozen biopsy material.

Antibodies to Helicobacter pylori in dyspeptic patients, asymptomatic adults, and children from Costa Rica

An enzyme immunoassay for the detection of serum IgG antibodies to H. pylori whole bacterial sonicate was used in a study of dyspeptic adult patients (n=165), apparently healthy blood donors (n=154), and children (n = 66) from Costa Rica, where a very high incidence of gastric carcinoma is observed. The mean antibody levels and frequency distributions were similar in the dyspeptic patients and the blood donors. Meanwhile lower antibody levels to H. pylori were more frequently observed in children than in adults, suggesting an age‐dependent increase in seroprevalence, as described in studies from other populations.