Pelayo Correa. Vanderbilt University. Nashville, Tennessee. US The Natural History of Gastric Cancer. Old and New Evidences.
Gastric cancer is the second most frequent cause of cancer deaths worldwide. Approximately one million cases are expected this year. Helicobacter pylori infection has been recognized as the primary cause of the disease but a very small fraction of infected subjects develop gastric cancer. The geographic distribution of gastric cancer and Helicobacter pylori infection contrast drastically. The so-called Africa enigma observed in several parts of the world is characterized by very high infection rates but very low cancer rates. This phenomenon emphasizes the classical epidemiologic model of causation in which the outcome of the infection is modulated by its interaction with host and environmental factors. The role of each set of factors will be analyzed. New evidence of the role of ancestral history of Homo sapiens and Helicobacter pylori in determining the cancer risk in Colombian populations will be presented. Although the mechanisms of gastric carcinogenesis are unknown, evidence will be presented on the possible role of epigenetic abnormalities.
___________________________________________________________________________________ STATE OF THE ART TOPICS I_______________________________________________________ E. R. Greenberg. Fred Hutchinson Cancer Research Center.Seattle, Washington USA The Challenge of Gastric Cancer in an Aging World.
Cancer now accounts for 7.5 million deaths per year globally, and two thirds of these deaths occur outside the high-income countries. Deaths from cancer will increase substantially over the next twenty years, with almost all of the increase occurring in low- and middle-income countries, largely a consequence of aging of their populations. Gastric cancer is now the 4th most-common cancer in the world (930,000 new cases), but it is 2nd only to lung cancer as a cause of cancer mortality (850,000 deaths). Although incidence and mortality rates for gastric cancer are declining in most countries, the number of cases and deaths is projected to increase; by 2030 roughly 1 million people will die annually from this condition, putting it in the top 10 causes of death worldwide. In many countries of Asia and Latin America, gastric cancer already is a leading cause of mortality. Despite its importance and the clear link to a treatable cause (Helicobacter pylori infection), programs to control gastric cancer have been limited and of uncertain effect. Except in Japan, where gastroscopy screening has become relatively widespread, gastric cancer has largely been ignored as a public health problem. Part of the reason why countries have not undertaken gastric cancer control programs is the lack of compelling evidence from controlled clinical trials that H. pylori eradication will decrease gastric cancer risk. Another issue is the perceived cost of wide-scale eradication programs. In 2009, the Southwest Oncology Group (SWOG) initiated a randomized trial in seven clinical centers in Latin America. The trial compares three different antibiotic regimens, employing inexpensive, locally available antibiotics, in terms of their effectiveness, cost, and acceptability. The long-term goal is to provide evidence that will lead either to a definitive trial of gastric cancer prevention through H. pylori eradication, or to the actual implementation of eradication
programs in adults without further clinical trials, based in part on the established benefits in peptic ulcer disease.
Steffen Backert. University College Dublin. Ireland Crosstalk Between the Type IV Secretion Effector Protein CagA and VacA Cytotoxin in Helicobacter pylori Signal Transduction
The development of gastric pathology induced by Helicobacter pylori infection is a long-term and multifactorial process depending on the host physiology and bacterial genotype. Epidemiological and functional studies have shown that virulent H. pylori strains carrying both the cag pathogenicity island (cagPAI) and the vacuolating cytotoxin VacA are key players in disease development. The cagPAI encodes a type-IV secretion system which forms a pilus for injection of the CagA effector protein into gastric epithelial cells. Injected CagA undergoes tyrosine phosphorylation by Src and Abl kinase oncoproteins. Phosphorylated and non-phosphorylated CagA can then induce actin-cytoskeletal rearrangements involved in host cell scattering and elongation, a process important in H. pylori-induced epithelial barrier disruption. We have recently reported that the CagA-induced responses can be inhibited in strains expressing highly active VacA cytotoxin. Our investigations revealed that VacA does not interfere with known activities of phosphorylated CagA such as inactivation of Src kinase and tyrosine-dephosphorylation of the actin-binding protein cortactin. Instead, we demonstrate that VacA exhibits inactivating activities on the epidermal growth factor receptor EGFR and HER2/Neu, and subsequently Erk1/2 MAP kinase which are important for cell scattering and elongation. Inactivation of the vacA gene, downregulation of the VacA receptor RPTP-alpha, addition of EGF or expression of constitutive-active MEK1 kinase restored the capability of H. pylori to induce the latter phenotypes. These data demonstrate that there is considerable crosstalk between the VacA and CagA virulence factors during infection. The data show that VacA does not only exhibit toxic cellular effects but can also downregulate CagA's effects on epithelial cells, a novel molecular mechanism showing how H. pylori can avoid excessive cellular damage. The latter model may explain how these bacteria live in a well-defined balance with its host in order to establish persistent infections in humans.
Douglas E Berg. PHYLO-GEOGRAPHY OF H. PYLORI GENOTYPES IN THE AMERICAS Dange Kersulyte1, Melissa Mendez1,2,3, Masato Suzuki4, Chihiro Sasakawa4, Awdhesh Kalia5, Thomas Boren3, Robert H Gilman2, and Douglas E Berg1
1 Washington University Medical School, St Louis, MO, USA, 2 Universidad Peruana Cayetano Heredia, Lima, Peru, 3 Umeå Universitet, Umeå, Sweden, 4 University of Tokyo, Tokyo, Japan, 5 University of Louisville, Louisville, KY, USA
BACKGROUND and ANALYSES: Helicobacter pylori is extraordinarily diverse genetically, with ≥3% sequence divergence on average among independent isolates, and different DNA sequence clusters predominating in separate human populations. These features reflect localized H. pylori transmission, historic separation of human populations, random genetic drift, and selection for locally adaptive traits. Many Latin American strains provided striking exceptions to the general correlations between H. pylori and human host ancestries. In particular, we found
strains from Lima region shantytowns to be mostly European in genotype, although most shantytown residents are originally from Andean Highlands and predominantly Amerindian in ancestry. In contrast, housekeeping gene sequences from H. pylori from a remote Peruvian Amazon community (Shimaa) were related to, but distinct from, those from East Asia. The genome of one representative Shimaa strain was sequenced completely. Shimaa strains contained cagA genes with novel EPIYA and CRPIA motifs, responsible for phosphorylation-dependent effects on cytoskeletal and tissue structure and phosphorylation-independent effects on transcriptional activation, respectively, and also novel allelesof hp0519, a gene thought to affect host tissue structure or function. In functional tests, Shimaa strains exhibited unusually weak activation of inflammatory and cell proliferative responses in cultured cell or Mongolian gerbil infection models, LeB (blood group O) specialist type BabA adherence to gastric epithelia, and difficulty in transformation with genes from unrelated strains. CONCLUSIONS. Shimaa H. pylori may descend from strains that infected the ancestral Asians who populated The Americas ≥15,000 years ago. Ancestral Amerind strains may have been less fit than and displaced by European strains in communities where both types circulated. Explanations for the apparently lower fitness of Amerind vs. European strains include (i) resistance to genetic exchange and adaptation to a varying environment; (ii) competitive ability, e.g., during mixed infection; and (iii) effects on host physiology, including human survival and efficiency of transmission.
Jay Solnick Catch me if you can: Modification of H. pylori Surface Proteins During Experimental Infection Cooke CL, Barrozo RM, Hansen LM, Styer CM, Huff, JL, Cariaga TA, Solnick JV Departments of Medicine and Microbiology & Immunology, Center for Comparative Medicine, University of California, Davis. Davis, CA. USA
Microorganisms commonly modify their surface proteins in order to adapt to changing conditions in the host, using a variety of mechanisms that include phase variation, antigenic variation, mutation, and even RNAi in some eukaryotic pathogens. Helicobacter pylori encodes a large family of paralogous outer membrane proteins (OMPs), the best studied of which is the BabA adhesin that binds fucosylated ABH antigens of the ABO blood group. We recently demonstrated that BabA expression is lost by antigenic variation and by phase variation during experimental infection of rhesus macaques with H. pylori J166. We now show that a similar phenomenon occurs in mice and in gerbils, though the mechanisms differ. Since BabA expression is lost in both wild type and RAG2-/- mice that lack functional B and T lymphocytes, adaptive immunity is probably not involved. We next turned to another surface protein, CagY, an unusual ortholog of VirB10 that is thought to coat the Type IV pilus and has extensive repetitive sequence motifs that are presumed to undergo antigenic variation under immune pressure. Induction of IL-8 requires CagY, but variant alleles are thought to maintain their function since this protein is essential for a functional cag pathogenicity island. To examine in vivo changes in IL-8 expression and allelic variants in cagY, we first challenged rhesus macaques with IL-8 inducing H. pylori strain J166. Beginning 8 weeks after challenge, strains recovered from infected macaques had reduced capacity to induce IL-8 compared to the input. These strains also had a change in cagY detected by PCR-RFLP and confirmed selectively by DNA sequencing. To determine if changes in IL-8 and in cagY were selected by the adaptive immune response, we infected C57BL/6 and immune deficient RAG2-/- mice with H. pylori J166. Beginning 8 weeks after challenge, H. pylori strains recovered from wild type mice showed a marked decrease in the induction of IL-8 that was accompanied by changes in cagY. In contrast, no changes were seen in IL-8 induction or in cagY in strains recovered from RAG2-/- mice. Alleles of cagY from
strains that retained the capacity to induce IL-8 complemented the ability of J166∆cagY to induce IL-8, while cagY alleles from strains that lost the ability to induce IL-8 did not. We propose that H. pylori harnesses the adaptive immune response to generate variants in CagY, which in turn yields a pool of strains with a modified inflammatory potential. These strains likely vary in fitness and allow H. pylori to adapt to changing conditions within the gastric environment. We hypothesize that modification of surface proteins during H. pylori infection is a mechanism to adapt to changing conditions of inflammation and glycan expression at the epithelial surface.
Francis Mégraud. Université Victor Segalen. Bordeaux France. Patterns of antimicrobial resistance of Helicobacter pylori and options for eradication regimens
Antimicrobial resistance is an evolving process which very much depends on the resistance mechanism. H. pylori resistance is essentially due to point mutations transmitted via a vertical transmission, and a steady increase in the resistance is observed following the selection pressure which depends upon the overall quantity of drugs used in a given country. Mechanism and prevalence: The most important resistance is macrolide resistance which is due to point mutations at certain nucleotide positions (2642 and 2643) on the domain V of the 23S rDNA. When the mutation is present, clarithromycin cannot bind anymore. A recent European survey carried out in 2008-2009 indicated a global resistance of 17% in adult strains with marked differences between those originating from northern countries and southern countries. Indeed, there is an excellent correlation between the macrolide consumption in a given country and the H. pylori resistance to macrolides indicating that the antibiotic consumption, notably for respiratory infections, is a major risk factor for resistance. Resistance to fluoroquinolones is an emerging problem because of the recent increase in the use of these drugs, and because some fluoroquinolones, especially levofloxacin, are now commonly given as second line therapy. The mechanism implies point mutations in the Quinolone Resistance Determining Region (QRDR) of the gyrA gene while a few cases may be related to an efflux mechanism. The prevalence in Europe is now close to 15%. Resistance to other antibiotics used for H. pylori infection, i.e. amoxicillin, tetracycline and rifabutin, does exist but is relatively rare. Metronidazole resistance is a special case. It is due to mutations in the nitroreductase gene rdxA and possibly in other genes, e.g. frxA, which play a role in transforming metronidazole into an active compound which breaks down DNA. In addition, the standard method of testing appears to be unreliable because of a lack of reproducibility and there is no correlation with the outcome of the treatment. Determination of susceptibility testing: Fortunately the detection of clarithromycin resistance can be made by molecular methods which provide a quick result. Among the numerous methods proposed, one is especially effective: a real-time PCR using a biprobe and the Fluorescence Resonance Energy Transfer (FRET) principle, followed by a melting curve analysis (MCA) on the amplicons. This method allows the detection of H. pylori and its clarithromycin resistance within 2 hours directly from the biopsy specimens. More recently a multiplex PCR followed by hybridization on a strip has been able to detect both macrolide and levofloxacin resistance (GenoType HelicoDR). Another method which does not require PCR is the Fluorescence In Situ Hybridization (FISH) which can be performed directly on a histological slide. The wide use of these methods should improve the management of H. pylori infection because it has been shown repeatedly that treatment given on the basis of susceptibility testing improves the results. Eradication regimens: In areas of high resistance, these tools should be used in order to tailor the treatment, i.e. if Clari susceptible, Clari + Amox should be prescribed, if Clari resistant Levo susceptible, Levo + Amox should be prescribed, if both Clari & Levo resistant, several options are possible, e.g. sequential treatment and bismuth- based quadruple therapy. Sander Veldhuyzen van Zanten Resistance Pattern and Options for Treatmen of Helicobacter in Canada Sander Veldhuyzen van Zanten and Karen Goodman. Division of Gastroenterology. Department of Medicine. 2-14A Zeidler Ledcor Centre. University of Alberta Edmonton, AB T6G 2X8 Over the years treatments to cure Helicobacter pylori infection have followed the same pattern as in the rest of the world. Indeed many randomized control trials have been conducted in Canada. The studies did establish triple therapy consisting of a proton pump inhibitor (PPI), clarithromycin and either amoxicillin or metronidazole as first-line therapy. Unfortunately the overall success rate with this therapy when used in the community is approximately 75%. Quadruple therapy consisting of a PPI, bismuth, metronidazole and tetracycline is the best tested and most robust second line therapy. It achieves cure in approximately 75% of patients. Resistance data from strains acquired across Canada show that resistance to metronidazole has remained relatively stable around 20%. In contrast resistance to clarithromycin has gone up from 2% in the early 90s to more recently 11-15%. Resistance to tetracycline and amoxicillin is rare. Limited data suggests that the background resistance to levofloxacin is high at 15%. A small recent RCT comparing standard triple therapy to the new "sequential" therapy showed that the latter is approximately 8% more successful.
In addition data will be shown about a unique Helicobacter project that has taken place in a small first nations community in the Northwest Territories, located close to the Beaufort Sea. The local community of Aklavik, consisting of 600 inhabitants, has expressed concern about the high rate of gastric cancer in their area. Based on results of urea breath test and endoscopically obtained biopsies the presence of Helicobacter in that community is approximately 60%. Histologically patient's have a much more severe gastritis than the average Canadian population.
Carlito B. Lebrilla. University of California, Davis. US Cancer Diagnosis with Protein Glycosylation
Proteins are often modified by the attachment of sugar units called glycans during the normal course of protein production. It is estimated that over 70% of all human proteins are modified in this way. The development of new analytical methods, specifically mass spectrometry and separation science, has significantly increased the progress in understanding the role of the glycome in many biological areas. Research in our group has focused on glycans as disease markers and their role in nutrition. For disease markers, a new paradigm for cancer biomarker discovery is proposed. The detection and analysis of the glycans that decorate the underlying polypeptide in glycoproteins may provide more specific detection of cancer rather than examining the proteins themselves. There are numerous studies that demonstrate glycans produced in cancer cells are different from those in normal cells. The aberration in glycosylation is observed with many types of diseases as well. In the studies in our laboratory, we harvest
glycoproteins and extract the glycans in patient serum to determine whether the glycosylation has changed in cancer patients compared to healthy patients. This new glycans assay is used to discover biomarkers for the diagnosis of gastric, breast, prostate, and ovarian cancer.
___________________________________________________________________________________ STATE OF THE ART TOPICS II______________________________________________________
Rolando Herrero. Fundación INCIENSA, Costa Rica Gastric Cancer in Latin America: the Contrasting Rates
Nearly 70,000 new cases of stomach cancer occur every year in Latin America and the Caribbean, with an estimated 55,000 deaths. Incidence and mortality are consistently higher in males than in females. Age –standardized rates show a steep increase with age, and despite declining rates in recent years, the absolute number of cases is likely to continue increasing with ageing of the population. Incidence and mortality rates of stomach cancer in Latin America present wide variations by country, with very high or high rates in Chile (46.1/100000 in males), Costa Rica, Ecuador, Peru and Colombia, and much lower rates in Mexico (13.1/100000 in males), Argentina, Uruguay and several Central American countries. Regional variation is also evident within countries, with ample differences by region, and higher rates in the mountain areas compared with low altitude areas. There are limited data to explain the vast differences in incidence and mortality from stomach cancer in Latin America, but they seem unlikely to be explained by prevalence of helicobacter pylori infection. Research is needed to determine if bacterial (HP strains), host (genetics, immune responses) or environmental (diet, salt, tobacco, other infectious agents, pesticides) factors explain the contrasting rates. These explanations could help develop preventive interventions against this cancer that remains one of the most common in many areas of Latin America. Incidence and mortality from stomach cancer by region in Latin America, males (Globocan 2002)
Incidence and mortality from stomach cancer by region in Latin America, females (Globocan 2002)
Martin J. Blazer. New York University. New York, NY. US The Discovery of CagA and Early Developments
Guillermo Perez and I begun studying the antigens of the “Gastric Campylobacter-Like Organisms” (GLCO) in 1985. Development of a serologic test for “C. pylori” identified individuals who had high-titer IgG antibodies to the organism. In 1988, my laboratory had begun cloning genes from Campylobacter species, using protein expression in the
bacteriophage λ gt11. As work about C. pylori was becoming increasingly interesting, we decided to try to clone antigenic proteins from that organism. In 1989, we screened a library of C. pylori genes in λ gt11, using serum antibodies from a person who had been identified as a carrier of the organism. Screening of the library identified several clones that were strongly recognized. The first clone purified (7-2/1) expressed an IPTG-inducible fusion protein with molecular mass >106,000 daltons that was first recognized by the human serum on July 20, 1989. Subsequent analysis by Murali Tummuru eventually described the cagA sequence. Nearly simultaneous studies in the lab conducted by Timothy Cover identified a 128 kDa protein in culture supernatants that was present in 60% of patients with gastritis alone and nearly 100% in patients with peptic ulceration; that protein also was CagA. Nearly identical proportions of ulcer and gastritis patients to those we observed were reported about 2 years later by Jean Crabtree and colleagues who described a “120 kDa” protein recognized by gastric IgA; their finding confirmed to us that we were on the right track. Within a few years, from our work and that of others (notably Jean Crabtree), it became clear that carrying cagA+ strains was associated with increased levels of gastric inflammatory cytokines and cells, and increased risk for both peptic ulcer disease and gastric cancer. From the early pioneering work of Antonello Covacci, Rino Rappuoli, Douglas Berg, and others came the realization that cagA and the cag genomic island on which it sits, are unique genetic elements. In 1995, Murali Tummuru reported that a gene near cagA, which we called picB was a strong homolog of type IV secretion system (TFSS) proteins, and that its mutation abrogated increased cytokine production by gastric epithelial cells. However, it was not until 2000 that several groups of investigators independently confirmed the functionality of the TFSS and showed that it was injecting the CagA protein into epithelial cells! In the past decade, the interest in CagA and the cag island has continued to grow, progressively uncovering the signaling of human cells, as well as the relationship of cag+ strains to both human disease and health.
___________________________________________________________________________________ STATE OF THE ART TOPICS III_____________________________________________________
Martin J. Blazer. New York University. New York, NY. US The Role of Helicobacter pylori in the Regulation of the Physiology of Children
There is substantial evidence that H. pylori has colonized the human stomach since time immemorial, and also much support for the concept that it has been dominant in this niche. H. pylori is highly interactive with the host, affecting epithelial, lymphoid, and neuroendocrine cells and pathways. For an ancient, dominant, interactive member of the human microbiota that is acquired early in life, as is H. pylori, one hypothesis is that there has been selection for microbes and hosts that can co-exist. However, over the course of the 20th, and now 21st centuries, H. pylori has been progressively disappearing in developed countries. As such, we can measure the consequences of the presence or absence of the organism. The presence of H. pylori over several decades of life is clearly associated with the risk of both peptic ulcer disease and gastric cancer, illnesses that overwhelmingly occur in middle and late stages of life. However, there has been much less investigation of the biological and clinical roles of H. pylori early in life. We know that H. pylori elicits host inflammatory and immune responses, and affects the local populations of microbes as well. There is evidence that H. pylori may provide partial protection against infectious diseases, particularly diarrheal illnesses which remain as an important cause
of childhood mortality, as well as tuberculosis. The absence of H. pylori also selects for different host T-cell populations, and a growing body of evidence provides support for the hypothesis that H. pylori presence may provide partial protection from asthma, as well as reactivation of tuberculosis. Finally, the stomach produces two hormones –leptin and ghrelin- that are centrally involved in energy homeostasis. There is increasing evidence that H. pylori is involved in their regulation, possibly through the action of local inflammation and cytokines. In consequence, a generation of children are now developing in the absence of our ancient gastric microbiota. The consequences of this change in human microecology are unknown, but may be fueling the explosive increases in obesity and diabetes that have occurred in developed countries in recent years. Inferences about the deleterious roles of H. pylori to the health of adults may not be applicable to its effects on the physiology of growing children.
Francis Mégraud.Université Victor Segalen. Bordeaux France. Prevention of gastric cancer by eradication of Helicobacter pylori
The discovery of Helicobacter pylori in 1982 has been a breakthrough in the field of gastroenterology. After proving its causal role for peptic ulcer disease, data have been gathered to show a causal association between H. pylori infection and gastric cancer. This relationship was already recognized by the International Agency for Research on Cancer in 1994, based on epidemiological data, and later an animal model using Mongolian gerbils was described. The limited proportion of H. pylori-infected subjects who develop a gastric adenocarcinoma can be explained by host factors (IL-Iβ polymorphism) and environmental factors (salty diet, lack of fruit and vegetables), but bacterial factors (cag positive strains) appear to be even more important. This finding has raised the question of eradicating H. pylori to prevent gastric cancer. The tools needed to achieve this goal are now available. H. pylori infection can be detected by a non-invasive test. Several tests have been described: the most accurate is the 13C-urea breath test, although the stool antigen test or serology can also be used with reasonable accuracy. Indeed, serology is the best suited method for detection because of the convenience. H. pylori infection can be cured by a one-week antibiotic regimen. The regimen recommended as the first intention therapy includes 2 antibiotics (clarithromycin and metronidazole or amoxicillin) with a proton pump inhibitor, which has an adjuvant role for increasing the stomach pH. It allows the eradication of H. pylori in 70 to 80% of the cases and a second intention therapy can increase the success to more than 90%. While detection and eradication of H. pylori are possible, questions have been raised concerning their use for prevention of gastric cancer in the population. In terms of efficacy, the main question concerns the existence of a “point of no return” for the evolution of gastric adenocarcinoma. The cancer occurs after several decades of infection during which, at least for the intestinal type, the mucosa reaches pre-malignant stages. An intervention study showed that the benefit of eradication on cancer prevention was only significant for subjects without pre- malignant lesions. Indeed, several studies have shown that a premalignant lesion intestinal metaplasia may not regress. With regard to safety, in addition to the limits existing in all prevention programs, there is a special threat concerning a selection of antibiotic resistance in H. pylori as well as in other bacterial species following an extensive use of these drugs. For these reasons and despite favourable criteria when gastric cancer prevention is compared to colon cancer prevention, the time for implementing a massive eradication of H. pylori for primary prevention of gastric cancer has not yet arrived. The age for intervention should be determined and a treatment free of adverse events, e.g. a therapeutic vaccine, should be developed. Lizbeth López Carrillo Helicobacter pylori, Nutrition and Smoking Interactions: Their Impact in Gastric Carcinogenesis
Lizbeth López-Carrillo1, Carlos A. González 2 1 Mexico National Institute of Public Health 2. Catalan Institute of Oncology Gastric cancer (GC) is the result of a long multi-step and multifactorial process involving possible interactions between Helicobacter pylori (H. Pylori ) infection, environmental exposures (diet, smoking and/or xenobiotics) and host genetic susceptibility. Interactions between H. pylori infection, tobacco smoking and dietary antioxidants are biologically plausible. Although relatively few epidemiological studies have evaluated the effect of the interaction between smoking and H. pylori infection on GC risk, there is a suggestion of a positive interaction between the two factors. In contrast, evidence suggests a negative interaction between dietary antioxidants and H. pylori infections on GC risk. The potential protective effect of dietary antioxidants such as vitamins C and E and b-carotene seems to be stronger in those infected by H. pylori, even though results are inconsistent. In Asian populations, subjects infected by H. pylori and with high dietary salt intake may have a higher risk of GC than subjects without H. pylori infection and with a low salt intake. The risk of GC associated with red meat, processed meat or endogenous formation of nitrosamines appears to only be observed in subjects infected by H. pylori. Capsaicin intake, the pungent principle of chile pepper, significantly increases GC risk only among subjects infected by more virulent H pylori cagA positive strains. Positive interactions between GC risk factors imply that, in certain subgroups of the population, the risk of GC associated with simultaneous exposure to these factors is higher than that in the rest of the population. These high risks subgroups should be the target for preventive measures. Identification of foods, such as yogurt, that may protect against H pylori infection, deserve attention to be assessed in further dietary intervention trials.
Keith T. Wilson.Vanderbilt University. Nashville Tennessee. US A Novel Mechanism for Oxidative Stress and H. pylori-Associated Gastric Cancer Risk in Colombia
Our laboratory has identified a novel pathway for production of oxidative stress in gastric epithelial cells exposed to H. pylori. The polyamine spermine is metabolized by spermine oxidase (SMO), which produces hydrogen peroxide (H2O2) that causes oxidative DNA damage.
In Colombia, regions of high gastric cancer risk in the Andean mountains and low cancer risk in the coastal areas have been identified. We determined if SMO-derived oxidative stress and H. pylori strain differences may contribute to cancer risk. In an analysis of 212 male subjects between ages 40-59, we found that the 25-fold difference in cancer risk could not be explained by the frequency of
cagA-positive strains. We used cagA vacAs1m1 strains, and randomly
selected 10 from each region for study. Strains from the high risk region induced significantly more SMO mRNA and protein expression, H2O2 generation, and oxidative DNA damage when
co-cultured with gastric epithelial cells in vitro. This DNA damage was attenuated when SMO was blocked with small interfering RNA. Immunostaining of gastric biopsies from the source
patients for these strains demonstrated increased SMO protein levels in gastric epithelial cells from high risk subjects that correlated with the oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine, and these findings were confirmed by flow cytometry. A gerbil-adapted high risk strain induced significantly more dysplasia, carcinoma, SMO expression, and DNA damage in gerbils than a low risk stain. Phylogenetic analysis of clinical isolates using multi-locus sequence typing identified that high risk strains were of European origin and low risk strains were of African and European origin, and that European strains were associated with more advanced histologic lesions. The relationship between phylogenetic strain origin and oxidative stress-induced DNA damage is currently under investigation. H. pylori strain differences appear to have an important role in generating oxidative stress and gastric cancer risk in Colombia.
WORKSHOPS: ___________________________________________________________________________________ WORKSHOP 1 RECENT EPIDEMIOLOGICAL TRENDS______________________
Luis Eduardo Bravo, Pelayo Correa. Colombia Trends in Cancer Incidence in Cali, Colombia
The cancer population-based registry in Cali began in 1962. It is located and sponsored by the Departamento de Patología de la Facultad de Medicina de la Universidad del Valle (Pathology Department of the Medicine Faculty of the Universidad el Valle). It is the only source with current data on cancer incidence in Latin America during the last 48 years. In time, the following trends have been observed: 1. Gastric cancer shows a decreasing rate in both genders, especially in men. 2. Incidence in cervical cancer has drastically decreased. 3. Incidence in colorectal cancer has gradually increased both in women and men. 4. Incidence in lung cancer gradually increased between 1962 and 1980. Since then, it has slightly decreased in men and has kept at the same rate in women. 5. Incidence in breast cancer gradually increased between 1962 and 1995. Since then, there has been a marked increase. Mortality rate has remained stable. 6. Incidence in prostate cancer gradually increased between 1962 and 1990. Since then, there has been a marked increase. Mortality rate has remained stable. The possible reasons of these trends will be discussed. Catterina Ferreccio. UCC Chile Recent Epidemiological Trends of H. pylori Infection and GC in Chile: Role of Socioeconomic Development.
The first studies of H pylori (HP) in the Chilean general population, published between 1990 and 1993, reported seroprevalence of 5% under 1 year of age, 75% at age 40, reaching 90% at age 50-60, to decline slightly at older ages. The National Health Survey of 2003, a population-based sample of people over 17 years old, showed a similar age curve reaching 78.6% at 50-60 years; ie, a 10% decrease in HP infection in a decade. Information about HP distribution and trends is scarce in contrast with the abundance and good quality of statistics about its main health effects i.e. gastric cancer (GC), which will be the focus of my talk. Chile is among the top five countries worldwide in terms of GC risk. Between 1960 and 1980, GC death rate per 100,000 populations decreased steadily from 40 to 20, associated with socio-
economic development. Nevertheless, in the last 30 years, GC mortality has not decreased further even though and important socioeconomic growth in the period. This lack of impact may be explained in part by the latency in the development of GC, but also by the huge disparity in income distribution in Chile. The recent economic growth mainly benefited a group which already was at lower risk of GC. The marked spatial structure in GC risk is significantly associated with poverty with GC Mortality rates varying from 5 to more than 50 between rich and poor counties. GC differential has also been evident in the only two cancer registries available in Chile, GC standardized incidence rate in the richer region of Antofagasta (7.4 and 18.5 women and men respectively) is half the risk of the poorer region of Valdivia (17.1 and 40.5 women and men respectively). Along with GC, HP seroprevalence was lower in Antofagasta (50.0 % and 66.7 % women and men respectively) than Valdivia (69.2% and 83.3% women and men respectively). GC structure reflects the structure of HP infection in the population. Thus counties with high, medium and low risk of GC presented average HP serological infection rates (%) of 79.7. 70.7 and 62.3 respectively (statistically significant test for trend); being much higher the difference at younger ages, thus at ages 17-24 years corresponding HP seroprevalence were 79.7%, 49.3% and 39.8%. In multivariate analysis HP infection is the main explanatory variable of GC distribution. We will discuss current efforts to identify suitable public health strategies for primary and secondary prevention of GC in Chile.
Dra. María Eugenia Cavazza Porro Epidemiology of Helicobacter pylori in Venezuela
Dra. María Eugenia Cavazza Porro. Laboratorio de Microbiología Molecular. Instituto de Biomedicina. Ministerio para el Poder Popular para la Salud. Universidad Central de Venezuela. [email protected]
In Venezuela, gastric cancer occupies third place among deaths caused by cancer in both genders (raw rate: 6.48% - specific rate 83.67%), and there is a raw rate of 8.07% in incidence. During the last years there has been a decrease in mortality rates, but it is due to the under-registration of cases since 2005. The distribution according to the types of cancer in the state of Táchira, the one with the highest mortality rate by gastric cancer, is: intestinal (64.5 %), diffuse (32.8%), and mixed (2.7%). In a preliminary analysis of 200 subjects in two different geographical areas: the state of Táchira (50% of cases) and Caracas (remaining 50%), the main lesions that were found were a Chronic Gastritis 56.74% and Chronic Atrophic Gastritis (15.35%). In general; in the Metropolitan group the most common lesion found was Diffuse Antral Gastritis. The Táchira group showed the same lesion but in a lesser percentage, and this group is showing an important increase in the frequency of Atrophic Chronic Gastritis (p=0.00001), type 1 metaplasia and dysplasia. A seroprevalence study in 370 symptomatic adults, 406 asymptomatic adults, 27 symptomatic children and 238 asymptomatic children showed a high seroprevalence of the infection, both, in the asymptomatic and the symptomatic groups. In the child population, the percentage of children with specific anti-H.pylory IgG antibodies varies between 30 to 60%. In the adult group the seroprevalnece varies between 68 to 93%, being the highest percentages in patients from the “Centro de Control de Cáncer Gástrico” (Gastric Cancer Control Center) of San Cristóbal, in the state of Táchira.
Dra. Beatriz Gutiérrez Carrillo. Instituto Finlay, La Habana, Cuba Gastric Cancer Epidemilogy: The Cuban case
A descriptive analysis of gastric cancer incidence was performed from the data granted by the Cancer National Register. Province/gender: with a worldwide population-adjusted rate / 100,000 inhabitants (PAR) for the city of La Habana, 7,8 male (M) and 5,1 female (F), increasing in Matanzas, Villa Clara, and Santiago de Cuba for M. Mortality/incidence/province/gender. Relative three-year changes 2001-2003 and 2004-2006 for Ciego de Vila 0,52, Guantánamo 0,60 gender F. S. Spiritus 0,34 and Isla de la Juventud 0,70 M gender. Mortality by gastric cancer/province/gender: corresponded the same as the former, with a higher PAR in the provinces of Pinar del Río, Villa Clara, Guantánamo and Santiago de Cuba for F gender 3,8 and M gender 4,3. Santiago de Cuba reports mortality rates of F gender 3 and M gender 3,2. Table 9. Incidence and mortality for gastric cancer in Cuba for the 2004-2006 period/groups of age starts at the age of 20 with an increase from 40 on up to 75 years of age for both genders. Incidence/clinical stages in both 3-year periods 2001-2003 and 2004-2006 for stage IV demonstrated differences between both groups with a bad prognosis. Quality indicators for both genders in both periods with no significant differences: HV (histological verification) DCO (only cases reported only by death certificate) W/S (without study). Distribution according to histological diagnosis Cuba/ Provincia Santiago de Cuba 2001- 2003: gastric adenocarcinomas 70/85 %. Santiago de Cuba, and in this province, the “Saturnino Lora” hospital reported 100 cases, a higher registration to the rest of the provinces and their hospitals. Karen J Goodman, University of Alberta, Edmonton, Alberta, Canada. H. pylori Infection and Gastric Cancer: Epidemiological Trends in Canada. Estimates of H. pylori prevalencein studies of average populations of Canadian adults range from 30-38%, decreasing over time in reports from 1994-2003; a 2005 study reported a prevalence of 5% in children from major urban centers. Elevated H. pylori prevalence occurs in Canadians who are elderly, immigrants from high-prevalence regions, or of Aboriginal ethnicity. Canada’s northern territories have high proportions of Aboriginal residents, and disease rates in these territories reflect health patterns of northern Aboriginal communities.Reports from 3 northern Aboriginal communities in Canada reveal H. pylori prevalence estimates ranging from 51-95% in adults and 32-56% in children, similar to reports from Alaska Native and Greenland Inuit communities. Gastric cancer rates are elevated in the northern territories compared to the rest of Canada, and though case counts for subgroups in this region are small, available data suggests this cancer is particularly frequent in northern Aboriginal groups. Recent community-driven research in northwestern Canada, conducted at the request of Aboriginal communities worried about health risks from H. pylori infection, show elevated prevalence of histopathologically diagnosed conditions of the gastric mucosa (severe inflammation, atrophy, intestinal metaplasia) that indicate increased risk of gastric cancer.
Rolando Herrero. Fundación INCIENSA. San José, Costa Rica Recent Epidemiological Trends: the Case of Costa Rica
Excluding skin cancer, stomach cancer in Costa Rica is the leading cancer in incidence among men and the third after breast and cervical cancer among women. It is the first cancer in mortality among men and recently became the second among women after breast cancer. Incidence and mortality have been declining in the last 15 years, with age-adjusted incidence rates among men of 43.0 per 100,000 in 1993 compared with 31.9 in 2003. Among women incidence has declined from 21 to 17 per 100,000 in the same period, with corresponding declines in mortality rates. Age-specific incidence and mortality rates show a steep increase, with a rate of 10.8 per 100,000 among subjects of both sexes at age 50-54 years compared to a rate 268.6 per 100,000 among the 75 years old and older (period 2000-2005). One of the most striking features of stomach cancer epidemiology are the regional differences, with a 5-fold variation between the high and the low incidence areas (55.0 per 100,000 in León Cortés vs. 12.0 in Abangares). Some areas in the mountains surrounding the Central Valley consistently present higher incidences, for poorly understood reasons that warrant investigation.
Dra. Cinthia Goldman. School of Pharmacy and Biochemistry, University of Buenos Aires, Arg. Recent Epidemiological Trends: The Case of Argentina.
Argentina has a population of 40 million inhabitants, with a 33% demographic concentration in Gran Buenos Aires. It is classified by the World Bank as an emerging economy. Six epidemiological studies were performed to determine the prevalence of Helicobacter pylori infection within the country and its associated risk factors. Two studies carried out in 1996 [Olmos et al. (2000); J Clin Gastroenterol 31(1):33-7)] and 1997 [Pest et al. (1999); Acta Gastroenterol Latinoam 29(5):297-305] which included 754 and 645 volunteers from around the country, reported 49.1% and 55.9% seroprevalence in adults, respectively. Seroprevalence was 15.7% for the pediatric group. Another multicentric study performed in 435 adults from Santa Fé in 2000, reported coincident results: 52.2% carried H. pylori antibodies [Jiménez et al. (2004); Acta Gastroenterol Latinoam 34(1):16-20]. In San Luis, instead, seroprevalence in 509 volunteers was significantly higher: 75.9% and 48.8% for asymptomatic adults and children [Mattana et al. (2004); Enferm Infecc Microbiol Clin 22(4):227-9]. In 2002-2004, our group evaluated 395 children with upper gastrointestinal symptoms from the Province of Buenos Aires by the 13C-Urea Breath Test, finding an H. pylori prevalence of 40.0% [Goldman et al. (2006); World J Gastroenterol 12(33):5384-88]. Prevalence was 24.3% for a similar population in 2006-2008 [Janjetic et al. (2009); J Pediatr Gastroenterol Nutr (in press)]. Overall, risk factors associated with the infection reported in the different studies were: age, socioeconomic status, sanitary standards, family crowding and educational level. Our analysis of 1025 children from Buenos Aires indicates a significant reduction in the prevalence of H. pylori from 40% in 2002 to 25% in 2009, a worldwide epidemiologic trend. The described reports suggest that epidemiological pattern of the infection in Argentina is similar to that reported in developed countries, with a prevalence variation within different regions.
Michael G. Bruce. Arctic Investigations Program, National Center for Preparedness, Detection and Control of Infectious Diseases, CDC, Anchorage, Alaska Recent Trends in Helicobacter pylori Epidemiology among Alaska Native Persons Background:H. pylori infection is common among Alaska Native persons (AN), is a major cause of stomach ulcers, and may be associated with stomach cancer. Methods: I will present data from
current and previous H. pylori studies conducted in Alaska, and data from Alaska state-wide surveillance looking at seroprevalence, antimicrobial resistance, treatment failure, reinfection and gastric cancer. Results: Seroprevalence was higher among AN (75%) compared with non-Natives (24%). Surveillance data demonstrated that 30% of H. pylori isolates were resistant to the antibiotic clarithromycin, 42% to metronidazole and 19% to levofloxacin. Rates of reinfection (2 years after successful cure) were highest among AN living in remote, rural villages (22%) compared to AN living in urban settings (14.5%) and non-Natives living in urban settings (12%). Among persons treated with clarithromycin-based therapy, treatment failed in 72% of persons carrying clarithromycin-resistant H. pylori. Among persons treated with metronidazole-based therapy, treatment failed in 19% of persons carrying metronidazole-resistant H. pylori. Gastric cancer incidence rates among AN are ~23 cases per 100,000 persons which is 3-4 times higher than in US Caucasians. Studies currently looking at the relationship between infection with H. pylori and gastric cancer are ongoing in Alaska. Conclusions: Prevalence of H. pylori infection is high among AN. A high proportion of H. pylori isolates demonstrate resistance to the key antibiotics used to treat this infection. Clarithromycin resistance is associated with a greater risk for failure with clarithromycin-based treatments. Among 3 different population groups in Alaska, reinfection after successful treatment for H. pylori infection occurred at rates higher than those reported for other US populations and were higherst among rural ANs. Gastric cancer incidence rates among AN are 3-4 times higher than in US Caucasians.
Douglas R Morgan.
SD Crockett*, JL Lund*, RL Dominguez**, DR Morgan*. *Division of Gastroenterology and Hepatology, University of North Carolina **Western Regional Hospital, Santa Rosa de Copan, Honduras
Use of an Endoscopy Database as a Gastrointestinal Cancer Registry Surrogate in a Resource-Limited Nation: Experience of the Western Honduras Gastric Cancer Project
Introduction: Cancer epidemiology is problematic in developing nations. Clinical experience suggests that the incidence of gastric cancer is high in Honduras, in contrast to the limited national statistics. The Honduras National Cancer Center (Emma Romero de Callejas) network of hospitals reports 112 total gastric cancer cases in the 7-year period from 1998-2004, the most recent statistics. IARC (Globocan 2002) calculates stomach cancer age-standardized incidence rates (ASRs) for Honduras of 15.2 and 10.8 per 106 person years (PY) for males and females, respectively, which are imputed from neighboring countries such as Costa Rica. The western region of Honduras provides a unique epidemiological niche to facilitate estimation of the incidence of gastric cancer for Honduras. This region, with a population of approximately one half million people, is well circumscribed by its geography (borders, mountain ranges) with a single district hospital, and established referral pattern. Methods: We conducted a retrospective analysis of a de-identified endoscopy database of incident cases of gastric cancer presenting to the Western Regional Hospital, Copán district, from 2002-2008. All patients who presented were symptomatic, as screening programs are absent in Honduras. Population data was obtained from the 2001 census, available from the national census institute (Instituto Nacional de Estadistica). ASRs were calculated with the world standard population for the five age classes of 0-14, 15-44, 45-54, 55-64, and over 65. Results: In the entire western region, there were 510 incident cases of gastric cancer during the 7-year study period. Standardized incidence rates were calculated, based upon the complete data was available for the immediate Copán district (Table 1). Mean annual incidence rates were
35.9 and 14.5 per 106 PY for males and females, respectively. Specific municipalities were shown to have significantly higher rates, and may be appropriate for targeted screening programs.
Conclusion: We have found that use of a comprehensive endoscopy database in a well-defined catchment area can serve as a surrogate for cancer registries in low resource nations. The true incidence of gastric cancer may be significantly higher given the lack of a screening program, the high percentage of advanced cases noted at the time of diagnosis, and the fact that a large percentage of the base population does not seek medical care. Gastric cancer in this low- resource area is associated with high morbidity and mortality, warranting both research and prevention programs. ___________________________________________________________________________________ WORKSHOP 2. BACTERIA GENETIC DIVERSITY AND VIRULENCE_______________
Javier Torres. Unidad de Investigacion en Enfermedades Infecciosas, IMSS, Mexico. The Case of Mexico.
Gene diversity in H. pylori is unusually large and polymorphisms are more frequent in virulence genes such as the cag pathogenicity island (cagPAI), vacA or adhesins. Sequence variation modify biologic activity and so, it is important for each population to learn variants prevalence in their group and determine the effect on virulence activity. We have studied a number of H. pylori strains isolated from Mexican patients with different gastroduodenal diseases and from different communities. We studied both, gene diversity and biologic activity of the different isolates. In the case of the 3´region of cagA, polymorphisms were very common, with EPIYA patterns of AC, ABC, ABCC, ABCCC or ABABC types. Isolates missing the B motif also lack activity on AGS cells. Otherwise, no clear association was found between the number and type of EPIYA motifs and the activity on cells. In contrast to previous reports, we found no association between the size of the 3´cagA region and disease. Next, we studied the gene content of strains from patients with non-atrophic gastritis, duodenal ulcer and cancer using whole-genome microarrays. We identified genes which were significantly less frequent in isolates from cancer patients, particularly genes involved in inflammation and in regulation of the response to acid, suggesting they are not Essentials at this stage of the infetion. In contrast, in strains from duodenal ulcer patients, genes involved in the response to acid were significantly more frequent. These results suggest that during years of interaction with a changing gastric mucosa, selective microenvironmental forces are acting on H. pyori populations to select those more fitted to adapt to the modified environment. We also studied the polymorphic regions of the cagA, babA and oipA virulence genes for sites under positive darwinian selection and found that sites under selection are inside regions important for biologic activity, such as the EPIYA motifs.
María Mercedes Bravo. Instituto Nacional de Cancerología, Bogotá, Colombia. Bacterial Genetic Diversity and Virulence. The Case of Colombia.
In Colombia there are clear and marked contrasts between infection by H. pylori and gastric cancer. The prevalence of H. pylori infection is high throughout the country; however general population living in the Andes Mountains has high rates of gastric cancer whereas those from the Atlantic or Pacific coasts have very low rates. Carriage of certain genotypes (cagA-positive, vacA type s1) is significantly associated with a higher risk of gastric cancer. The distributions of vacA and cagA genotypes have been evaluated in H. pylori cultures or gastric biopsies from patients from high and low gastric cancer risk regions. VacAs1a subtype has been found predominant in the central region of the country and also in the Atlantic coast (60% of all s1 strains), in contrast vacAs1b is the main subtype in the south of the country and also in the pacific coast (95% of all s1 strains), these findings suggest that regional variations in the dominant circulating strain occur. vacAs1m1 strains are predominant in all regions with prevalences ranging between 60 and 80%, vacAs2m2 genotype is observed in 7.3 to 29.2% of strains, s1m2 in 1 to 4.3% of strains. Prevalence of CagA positive strains ranked between 90,4% and 64,3%. Two studies have compared the distribution of H. pylori genotypes in regions with contrasting gastric cancer risks and similar rates of H. pylori infection. One of them compared the genotypes from the pacific coast (Tumaco) and two cities in the Andes mountains in the south of the country (Pasto and Túquerres). s1m1 strains were more common in the high risk area (88.3% vs. 67.4%) while s2m2 strain were more common in the low risk area (3.9% vs. 16.3%), these differences were statistically significant (p=0.044). Also the more virulent cagA positive vacA s1m1 strains were significantly more prevalent in the area with high risk than in the area with low risk (84.3% vs. 60.5%; p= 0.011). These differences could contribute to the differences in gastric cancer risk between those two regions. The second study contrasted genotypes from strains from the Atlantic coast (Barranquilla, Monteria, and Santa Marta) with genotypes from strains from the Andes Mountains in the central part of the country (Bogotá and Tunja). vacAs1m1 strains were more common in the low risk than in the high risk area (71.01% vs. 61.19 %) while vacAs2m2 strains were more common in the high risk than in the low risk area (32.8% vs.21.74%). In contrast cagA positive and cagAs1m1 strains were more frequent in the Atlantic coast than in the center of the country (70.3%, 54.15% vs. 63.2%, 53.5% respectively). The differences in cancer gastric risk between these two regions could not be explained by differences in the virulence of circulating strains. Several works have analyzed the distribution of genotypes among different patients groups (gastritis, atrophic gastritis, intestinal metaplasia and gastric cancer), in general genotypes s1, m1 and cagA and citotoxic strains cagAs1m1 are predominant in all diagnostic groups. The 3’ -end variable region of the cagA gene has also been analyzed in two studies. One of them analyzed strains from low (Tumaco) and high risk regions (Túquerres). A 64.2% of strains contained one EPIYA-C motif, 34.3% contained two EPIYA-C motifs and 1.5% contained three EPIYA-C motifs. Strains with one EPIYA-C motif were associated with less severe disease (non atrophic and atrophic gastritis) whereas strains with two or three EPIYA-C were associated with intestinal metaplasia and dysplasia (p<0.001). The distribution of strains with one, two or three EPIYA-C motifs was not significantly different between the two areas. In the second study 106 strains from Bogotá were analyzed, 62.3% contained one EPIYA-C, 31.3% contained two EPIYA-C and 0.9% contained three EPIYA-C. In spite of the similitude of EPIYA-C distributions with the Tumaco and Túquerres study, an association of a higher number of EPIYA-C motifs with a higher risk of gastric cancer was not observed in this study.
Diana Ortiz Princz.Instituto de Biomedicina (MPPS- UCV) Caracas-Venezuela Bacterial Genetic Diversity and Virulence: The Venezuelan Case
In Venezuela, H. pylori seroprevalence varies between adults from 70 to 90% while in children is around 35%. Stomach cancer is the first cause of death among men related to cancer and the third among women after cervix and breast cancer. Stomach cancer is the leading cause of cancer’s deaths In the Andin Region, being one of the areas with the highest risk worldwide. Biopsies from 50 patients from the Andin Region, state of Táchira, Venezuela, were evaluated using the Polymerase Chain Reaction, finding a 50% H. pylori positivity. 72% of the H. pylori positive individuals were cagA positive and from them 48% were vacAs1/m1, 29% vacAs1/m2 and 14% vacAs2/m2. In a group of 60 strains isolated from adult patients (49 ± 15 years) from a hospital in Caracas, 90% were cagA positive, 65% vacAs1/m1, 23% vacAs1/m2, 12% vacAs2/m2 and only 12% were babA positive, being the most predominant genotype cagA/vacAs1/m1 /babA negative. 85% of the pathological anatomy studies reflected the presence of moderate-to-severe chronic gastritis with intestinal metaplasia. 67 children from the Hospital Elías Toro, in Caracas; were also evaluated finding 35% positive to H. pylori. The genotype determined in 10 strains revealed the presence of the cagA gene in 100% of the isolated strains, being vacA s1/m1the predominant combination (90%). Importantly, the findings regarding the presence of the babaA gene in our populations have indicated a low positivity (30%). The distribution of all the genotypes studied were: 60% cagA+ vacAs1/m1 babA-, 30% cagA+ vacAs1+ babA2+ and 10% cagA+ vacAs2/m2 babA- . In Venezuela there is a high prevalence of H pylori infection among children and adults, plus a high percentage of strains presenting the cagA gene and more virulent vacA allelic variants preferably associated with the development of atrophic chronic gastritis and intestinal metaplasia, highlighting that the Andin area is a high prevalence gastric cancer region. The molecular diagnosis of H. pylori infection and characterization of the type of strain present in these high incidence areas are important to direct treatment an adequate follow up to the patient evolution.
Guillermo I. Perez Perez. New York University. New York, NY. US The Case of Native American Communities Helicobacter pylori is a Gram-negative bacterium which has been colonizing the gastric epithelium of modern humans since their migration from East Africa 58,000 years ago. H. pylori prevalence correlates with socio-economic status; Native American communities which have the lowest socio-economical level in the whole American continent are expected to have a high prevalence of H. pylori. Several studies in those communities have confirmed this high prevalence (>70%). The most accepted theory on the origins of the Native American populations is that they descended from East Asians that crossed the Bering Strait; thus, it was proposed that the genetic profile of H. pylori in Native American communities will be of East Asian type. However, most early studies in mixed populations (mestizos) showed that only the European genetic type was present. More recent studies targeted isolated communities of Native Americans have confirmed that the East Asian genotype is present in a small proportion of individuals in these communities. Our group, in collaboration with an international group of researchers, has confirmed the high prevalence of H. pylori among Native American populations, including the Northern Territories in Canada, individuals from an Apache reservation, and Amerindians from remote locations in Venezuela. A major discrepancy in the prevalence of CagA has been observed between the Native American communities that may require further studies. In addition, our group in collaboration with other investigators, has been studying in the genetic characteristics of H. pylori strains isolated from Native American populations. We have identified several genes that
differentiate East Asian from Western genotype including cagA, hspA, and vacA. We have studied strains from the Northern Territories, Mexico and Venezuela and confirmed that Native American strains are mainly Western type. In addition, some strains showed both western and Eastern characteristics, suggesting recombination occurred. These observations suggests the presence of ancestral Native American H. pylori strains but based on the proportion of those strains identified there is a tendency to disappear.
Vanesa Ramirez Genetic Diversity and Bacterial Virulence: “The Costa Rican case” Jerson Garita-Cambronero1; Rafaela Sierra1, Clas Une1, Wendy Malespín-Bendaña1, J.A. Ramírez2, A. de Mascarel3, R. Barahona2, R. Salas-Aguilar2, R. Páez2, G. Avendaño2, A. Ávalos2, Guillermo Pérez-Pérez4, Francis Mégraud3, & Vanessa Ramírez1.
1Instituto de Investigaciones en Salud (INISA), Universidad de Costa Rica (UCR), 2060, San José, Costa Rica.
2 Hospital Dr. Rafael Calderón Guardia, San José, Costa Rica.
3 Université Victor Segalen Bordeaux II, Bordeaux, France.
4 New York University School of Medicine, New York, USA
Studies concerning gastric cancer in Costa Rica started in the 1980s and continue today, being performed by multidisciplinary groups that belong to different public and private institutions, both national and foreign. Concerning the study of Helicobacter pylori infection and its association as a risk factor in gastric pathologies, diverse populations of dyspeptic patients from the Valle Central of Costa Rica have been analyzed. The prevalence of cagA positive H. pylori infections has been determined by serology in each of the studied populations, confirming that the serological detection of these strains is associated with the development of precancerous and cancerous lesions. In general, it has been determined that there is a high prevalence of the infection in the different populations evaluated. The first serological studies showed that there is no significant difference in the infection with this bacterium between areas of high and low incidence of gastric cancer nation wide, therefore, arising the necessity to establish the genotypic variability among the Costa Rican strains. Molecular studies have been developed in which the composition of the CagPAI and its associated gene expression have been evaluated, as well as the variability in the genetic plasticity zone in which the genes with potential to be risk markers are found. Furthermore, studies carried by private institutions in the country found a higher presence of H. pyloricagA, vacA s1m1 strains in regions of high prevalence of gastric cancer. Currently, the Program “Programa de Especial Interés Institucional Epidemiología del Cáncer, of the INISA-UCR” studies the risk of developing pathologies associated to the presence of factors: cagA; cagE; vacA s,m,i; babA2, oipA; phosphorylated EPIYAs in CagA, among others. Mariana Catalano.Universidad de Buenos Aires Argentina. The Case of Argentina. Helicobacter pylori Genetic Diversity in Individual Hosts Helicobacter pylori putative virulence factors can undergo a continuously evolving mechanism as an approach to bacterial adaptation to the host changing environment during chronic infection. cagPAI, oipA, vacA and dupA genes genetic diversity among 229 isolates obtained from multiple biopsies (niches) from the antrum and corpus of 40 patients was investigated. In addition, gene conversion of babA and babB in chromosomal loci: A (downstream of jhp0835),
B (downstream of jhp1165), and C (between jhp0298-jhp0301) was investigated in 100 isolates from 20 patients. lspA–glmM RFLP and RAPD-PCR evidenced mixed infection in 2/40 patients. cagPAI, oipA and dupA genes showed inter-niche variation in approximately 1/4 patients, conversely, mixed vacA-s-m alleles in a single host was a less common event (approximately 1/10 patients). Inter-niche variation of cagPAI genotypes in a single host included: diverse cagPAI deletions in different niches, variants with intact and with partially deleted islands, variants with empty-site-positive and with partially deleted cagPAI, or variants with an intact cagPAI and with empty-site-positive. Half of the patients with different cagPAI genotypes harboured an intact island in at least one niche. Inter-niches oipA CT repeat patterns difference included also ‘on’ and ‘off’ mixed patterns in single host. Inter-niche dupA differences involved the absence and presence of jhp0917 and/or jhp0918 or mutations in dupA, including those that may originate a non-functional gene. Inter-niches variation in gene conversion of babA and babB was observed among 13/20 patients. Locus A showed more stability that locus B, where genotype A/B was most frequently evidenced. Isolates from 5/20 patients showed at least in one niche two copies of babA located at different loci. From all the evolving markers studies, babA showed the highest inter-niches variability. Co-existence of diverse genotypes of putative virulence factors in a single host must be considered when drawing a correlation with clinical presentation.
Maria G. Domínguez-Bello. University of Puerto Rico, San Juan, PR, USA Diversity and Coevolution of H. pylori in Native Americans and Mestizos H. pylori is an ancestral inhabitant of the human stomach, a component of the human microbiota. The bacterium diverged with humans and adapted to specific characteristics. Selection occurs at the individual level, but also at the gene level, in which unequal recombination between strains favor an increasing prevalence of specific strain types. The whole genome of Amerindian H. pylori strain V225 shows distinctive features. While multi- protein phylogenetic trees reflect major human migration, host-interactive genes vacA and cagA show substantial divergence of Amerindian from Old World strains, and indicate new genotypes (e.g. VacA m3)involving these loci.
___________________________________________________________________________________ WORKSHOP 3. HOST FACTORS, POPULATION-SPECIFIC GENETIC RISK FACTORS____ Carmen Maldonado-Bernal The Case of Mexico Carmen Maldonado-Bernala,e, Alejandra Trejo-de la Oa, Martha Pérez-Rodríguezb, Margarita Camorlinga-Poncea, Lourdes Flores-Lunac, José M. Abdo-Francisd, Eduardo Lazcanoc, Javier Torresa.
aUnidad de Investigación en Enfermedades Infecciosas, Hospital de Pediatría, CMN SXXI, IMSS. bUnidad de Investigación en Inmunología, CMN SXXI, IMSS, Mexico City, México.
cCentro de Investigación en Salud Poblacional. Instituto Nacional de Salud Pública, Cuernavaca, Mor. dUnidad de Gastroenterología Médica, Hospital General de México. eHospital Infantil de México “Federico Gómez”. Mexico, Cyti, México.
Toll-like receptors (TLRs) participate in H. pylori recognition, and single-nucleotide polymorphisms (SNPs) in TLRs are associated with impaired immune response. We evaluate the association of TLR4/D299G/T399I, TLR5/R392*/N592S and TLR9/G2848A/T1237C SNPs with gastroduodenal diseases; and study the effect of SNPs on cytokine and chemokine expression in the gastric mucosa. Study included 450 Mexican patients with gastroduodenal diseases. SNPs in TLRs 2, 4, 5 and 9 genes were analyzed by allele-specific PCR. Cytokines and chemokines were assessed by qRT-PCR and immunoassay. TLR4/D299G/T399I polymorphisms were more frequent in duodenal ulcer and showed a trend in gastric cancer, when compared with non-atrophic gastritis. Patients with TLR4 polymorphisms expressed significantly lower levels of IL-1β, IL-6, IL-8 and GRO-α; and higher levels of TNF-α, IL-10, MCP-1 and MIP-1α. TLR5/R392* polymorphism was more frequent in cancer than in non-atrophic gastritis. Patients with TLR5 polymorphism expressed significantly lower levels of IL-1β, IL-6, TNF-α, and IL-10. However these patients expressed higher levels of IL-4. TLR9/G2848A polymorphism was more frequent in duodenal ulcer and TLR9/T1237C was more frequent in cancer, when compared with non-atrophic gastritis. Patients with TLR9/G2848A/T1237C polymorphisms expressed significantly lower levels of IL-1β and TNF-α. SNPs in TLRs 4, 5 and 9genes had an association with severe H. pylori associated disease and with modified pattern of inflammatory cytokines and chemokines in the gastric mucosa. These results suggest that TLRs 4, 5 and 9 SNPs contributes importantly to the clinical outcome of H. pylori infection. In other hand, Tumour Necrosis Factor (TNF) and Heat Shock Protein 70 (HSP70) are important molecules in inflammatory, infectious and tumoral processes. The genes codifying these molecules are polymorphic and certain alleles have been associated with susceptibility to disease. In this work was analyzed whether polymorphisms in inflammation-related genes are associated with the development of gastric cancer. The study included 447 Mexican adult patients, and 132 asymptomatic individuals. DNA was typed for the SNPs 2308 of TNF-α 1252 of TNF- , 1190 of HSP70-1, 11267 of HSP70-2 and 12437 of HSP70-HOM. Compared with the asymptomatic group, there was a significant association of TNF-β*A and HSP70-1*C alleles with gastric cancer (OR 5.69 and 3.76, respectively) and HSP70-1*C with duodenal ulcer (OR 3.08). Polymorphisms in TNF and HSP70 showed a significant severity-dose-response as risk markers from preneoplastic lesions to gastric cancer in Mexican population, probably because of their association with an intense and sustained inflammatory response.
Alejandro Corvalan. P.Universidad Catolica de Chile. Santiago de Chile The Case of Chile
In Chile, Gastric Cancer (GC) has the highest mortality rate in adults. Although population-specific risk factors have identified markers for gastric atrophy, the precursor lesion for GC, direct population-specific risk factors associated with GC development have not yet been found. Aberrant hypermethylation, an epigenetic mechanism of inactivation of tumor suppressor genes, is an emerging approach to identify such risk factors. We have tested aberrant hypermethylation by candidate gene approach in 32 cases of GC using Methylation Specific-Polymerase Chain Reaction (MS-PCR) approach. Seven (29%) out of 24 tested genes were aberrantly hypermethylated in at least 50% cases. These genes were next evaluated in tumor/plasma pair of 43 prospectively collected GC cases and compared with 31 age-matched non-cancer controls. Among these seven genes, only Reprimo, a repressor of G2/M transition dependant of p53 activation, was significantly aberrantly hypermethylated in plasma from GC cases vs control cases (p<0.001). Next, we prospectively collected another 23 GC patients from ambulatory center and 46 matched-control cases. Detection of aberrant hypermethylation of RPRM was identified in 20/23 (86.9%) and 20/23 (86.9%) of tumor and plasma from GC cases,
respectively by MSP. Next by Methylight, a quantitative realtime PCR assay, level of MyoD, a marker of cell-free DNA plasma was 36.4 copies/ml in control cases but 2,387.1 copies/ml in GC cases. These differences were statistically significant (p< 0.001 by Fisher’s exact test). Next we calculated the level of Reprimo after normalization by MyoD x 10,000. Levels of Reprimo were 0.19 and 7.49 in controls and GC patients respectively. These differences were statistically significant (p< 0.001 by Fisher’s exact test) and suggest that not only GC patients contain more cell free DNA in plasma but also that level of RPRM could be predictive of GC in control cases. Finally we performed endoscopy in 2 control cases in which RPRM levels were similar to that of GC. One case was normal and in the other chronic gastritis was found. In summary, our data indicate that we have identified a direct host factor (Reprimo) associated with GC. This host factor can be detected in tumor and plasma from GC cases and might be applied as a non-invasive screening method for GC in control cases.
Supported by FONDECYT grants 103033 and 1080563 and FONIS grant SA06I20019 from Government of Chile. Pending patent 000694 INAPI, Goverment of Chile.
María Correnti. Universidad Central de Venezuela. Caracas Venezuela. The Case of Venezuela.
Gastric cancer is one of the leading causes of cancer death worldwide. H. pylori (HP) infection increases the risk of developing a gastric precancerous condition, together with genetic and environmental factors and specific morbid conditions. In Venezuela, H. pylori prevalence reaches 95%. Recent studies performed in patients with precancerous lesions and HP infection in the state of Táchira, showed that other risk factors were a diet rich in starches and low in fresh fruits and juices, cigarette smoking, and a family background of the disease. Age presented a strong co-relation to advanced precancerous lesions; while an educational level had an inverse relation with the risk of metaplasia and dysplasia, as well as the use of refrigerators to preserve food. Overcrowded conditions had not relationship with the prevalence of premalignant lesions in this region. Other studies performed in the state of Monagas reported a co-relation between patients infected with Hp (76.38%) and overcrowded conditions, poverty, no access to public services (water and hygienic conditions) which favor fecal-oral contamination. In the metropolitan area, results indicated that around 74% of the persons with gastric lesions presented HP infection and that overcrowding was the main risk factor for infection. On the other hand, it has been reported a high prevalence of CagA-positive strains, between 50 to 85%, in different areas.
Clas Allan Une The case of Costa Rica
Clas Une, Wendy Malespín-Bendaña, Warner Alpízar-Alpízar, Jerson Garita-Cambronero, Rafaela Sierra & Vanessa Ramírez. Instituto de Investigaciones en Salud (INISA), Universidad de Costa Rica, 2060, San José, Costa Rica. The incidence and mortality rates for gastric cancer in Costa Rica are among the highest in the world. The genetic and environmental factors underlying this situation are poorly understood. The general population of Costa Rica has been reported by Morera et al. to be trihybrid, with
genes of European (61%), Amerindian (30%) and African (9%) origin, similar to those of other Latin American countries. The Program of Cancer Epidemiology at the University of Costa Rica is analyzing possible genetic risk markers for gastric cancer with a focus on inflammation regulatory interleukin genes. We have found a significant association of IL-1B+3954 and IL-1RN polymorphisms with gastric cancer. On the contrary, the IL-1B-31, IL-1B-511, IL-10-592, IL-10-819 and p53-codon 72 polymorphisms were not significantly related to gastric cancer. None of the studied polymorphisms were associated with dyspepsia. These data were partially confirmed by Con et al. who found an association between gastric cancer and IL-1B+3954 and IL1-1RN polymorphisms but also with IL-10-592 A-carriers. Serum pepsinogen I (PGI) concentrations and the PGI/PII ratio were associated with gastric cancer and atrophic gastritis but not with IL-1B or IL-1RN polymorphisms. We are currently analyzing the frequency of different markers in a population selected to be representative of the population over 50 years in Costa Rica in order to see what proportion of the population would be considered at risk using different combinations of risk markers. This population consists of 2600 persons who participate in a broad survey of ageing (CRELES). The allele frequencies for IL-1B+3954 and IL-1RN were similar those reported in Caucasian populations. The frequencies for IL-1B-31, IL-1B-511 and the IL-10 polymorphisms were, however, more close to those described in East Asian populations. Present and future work includes the study of polymorphisms of other genes, e.g. IL-8, TNF-α, TGF-β and IFN-γR and gastric pathology.
___________________________________________________________________________________ WORKSHOP 4. CHILDREN, THE STORY BEGINS____________________ Michael G. Bruce. Arctic Investigations Program, National Center for Preparedness, Detection and Control of Infectious Diseases, CDC, Anchorage, Alaska Epidemiology of Helicobacter pylori infection among Alaska Native Children
Background:H. pylori infection is common among Alaska Native (AN) children, is a major cause of stomach ulcers, and has been demonstrated to be associated with stomach cancer. Methods: I will present data from H. pylori studies in children conducted in Alaska and across the Arctic countries looking at prevalence, antimicrobial resistance, treatment failure, peptic ulcer, disease, iron deficiency anemia, and gastric cancer. Results: Seroprevalence was very high among AN, Greenlandic, and Northern Canadian children from a young age (16% acquisition per year in N Canada). Data from Alaska sentinel surveillance demonstrated that 17% of H. pylori isolates from children were resistant to the antibiotic clarithromycin and 33% to metronidazole. In a study performed in Greenland, 68% of children failed initial treatment with amoxicillin, metronidazole and a proton pump inhibitor. Rates of reinfection (40 months after successful cure) among Alaska Native children 7-11 years of age were high at 52%. Rates of peptic ulcer disease (all age groups) were elevated in Alaska (299/100,00) and Northern Canada (394/100,000). Gastric cancer incidence rates (all age groups) among Indigenous people residing in Alaska, Northern Canada, Greenland and Sweden were 2-4 times higher than those of non-Indigenous people. Anemia and iron deficiency affect up to 40% of children across the Arctic countries. Conclusions: Prevalence of H. pylori infection is high among AN children. A high proportion of H. pylori isolates from AN children demonstrate resistance to the key antibiotics used to treat this infection. Reinfection in Alaska children after successful treatment for H. pylori infection occurred at rates higher than those reported for other populations.
Karen J Goodman, University of Alberta, Edmonton, Alberta, Canada. What We Know about How We Get Helicobacter pylori Infection: the Importance of Being a Child. Initial infection with H.pylori occurs most frequently in childhood, with high incidence rates among children in the developing world and some high-risk groups in developed countries. Chronic H.pylori infection is associated with residential crowding and having H. pylori-positive family members. Key questions about H.pylori transmission remain unanswered due to research challenges stemming from uncertain accuracy of methods for detecting H. pylori in young children,difficulty in detecting new cases at onset, unknown frequency of transient infection,and bacterial characteristics that complicate isolation and identification of strains. Evidence suggests that H.pyloriusually spreads directly from person to person; the relative importance of fecal-oral, oral-oral or gastric-oral pathways is unclear, however,as is the role ofwaterborne or zoonotictransmission. A systematic review identified 37 cohort studies of H.pylori infection in children, with detection by urea breath test in 10, stool antigen test in 11, serology alone in 14 (8 retrospective), serology and eitherUBT or SAT in 5, other methods in 2.Serology results from 0- 12 months of age were excluded due to potential confusion with maternal antibodies. The studies consistently show incidence highest in the first two years of life.Spontaneous elimination frequencies were reported for 5 UBT studies, 6 SAT studies, and 7 serology studies, all but 1 showing evidence of transient infection. Follow-up intervalsand investigated risk factors varied greatly across studies, thus there is no solid body of evidence onacquisition and elimination rates across ages in childhood or factors associated with acquisition or elimination of H.pylori.
Margarita Camorlinga Inflammation Associated to Helicobacter pylori in Mexican Children Margarita Camorlinga1, Leopoldo Muñoz1, Francisco Aviles1, Armando Madrazo2, Sara Huerta4, Carmen Maldonado4,Guillermo Ramón3, Javier Torres1. UIMEIP. IMSS1. Servicio de Gastroenterologia2, Servicio de Patología3, IMSS. Hospital Infantil4. México. Helicobacter pylori (Hp) infection starts in childhood and persists for the life of the infected individual, manifesting non-atrophic gastritis, peptic ulcer, gastric atrophy or cancer in adulthood. The characteristics and consequences of the infection in children are not well known; but when HP is acquired very early in life, the risk for gastric cancer increases. Children also develop chronic gastritis and gastric ulcers although it is not frequent. A subgroup of infected children presents recurrent abdominal pain (RAP), however a causal association between both has not been demonstrated. To learn more about the characteristics and consequences of the infection in children, we have studied 731 children with RAP since 1995, with an average of 9.5±3.9 years of age. 22.6% of these children were infected with HP. Gastric biopsies, cellular proliferation and cellular and innate immune responses were studied by immunohistochemistry and morphometric techniques. Hp+ children presented a moderated-to-severe presence of mononuclear cells and neutrophils when compared with Hp- children, who showed a non-existing-to moderate infiltration. IL-8 levels, epithelial proliferation; macrophage, T and B lymphocyte infiltration were significantly higher in Hp+ children than in non-infected children. The epithelial proliferation was associated to the infiltration of polymorphonuclear cells but not with lymphocyte infiltration. The presence of Hp significantly decreased TLRs 2 and 4 and increased TLR5 expression.
In conclusion, Mexican children respond to HP infection with a low inflammatory response: an increase in T and B cells, increase in cellular proliferation and IL-8 expression, and a decrease in 2 and 4 TLRs’ expression.
Ximena Duque. Unidad de Investigacion en Enfermedades Infecciosas, IMSS, Mexico. H. pylori Infection,Growth, Anemia, and other Stories Ximena Duque, Jenny Vilchis, Robertino Mera, Segundo Morán, Fabiola Navarro, Ma. Eugenia Mendoza, Javier Torres, Pelayo Correa. Backgrounds:H. pylori infection is highly prevalent in developing countries and mainly acquired during childhood; however, its effects in children’s health are not well known yet. General objective: To evaluate the effect of H. pylori infection in the nutritional status in low socio-economic level school populations in Mexico City. Participants and methods: Students from boarding schools (n=941), age 6-13. Anthropometric weight and height measurements were performed in the beginning of the study; blood samples were taken to evaluate micronutrients and a breath tests with 13C-urea for diagnosis of H. pylori infection. A questionnaire on their sociodemographic conditions was answered by the parents or tutors. During the follow up, breath tests were performed every 6 months. Another group of 701 students from public schools in Mexico City were studied; blood samples were taken to evaluate the nutritional iron status. From these, 119 children (17%) presented iron deficiency and/or anemia and to them breath tests with 13C-urea were applied for the diagnosis of active H. pylori infection. H. pylori infected students (n=38) received eradication therapy and were randomly chosen to receive iron supplements (30 mg daily per 3 months) as ferrous sulfate or a placebo. Students without infection were also randomly assigned and received the same supplementation scheme with ferrous sulfate or other supplement (polymaltose iron, group not included in this report). After supplementation, blood samples were taken to evaluate the effect of the different treatments. Students in whom the bacterium was eradicated, were included in the analysis. Prevalence, incidence rates and spontaneous reversion rates to H. pylori infection were estimated. The association between H. pylori and height was explored and the effect of the eradication of H. pylori and/or iron supplementation on the iron nutritional status in students with iron deficiency was evaluated. Results. Prevalence, Incidence, reversion: The prevalence of active H. pylori infection in the studied population was of 37.9% (IC95% 34.8 a 41.1). The average duration of the follow up was 3 years, during which a 6.6 case incidence and a spontaneous reversion rate of 9 cases per 100 was estimated in children followed up per year. Association between H. pylori infection and height:H. pylori infected children were in average 1.32 cm shorter that the children without the infection (IC95% -0.42 a -2.22). This association was modified by age: the height of the children with the infection was 0.66 cm less for every year older (IC 95% -0.15 a -1.17). Effect of H. pylori eradication and iron supplementation in the nutritional iron status of children with iron deficiency. After H. pylori eradication children showed a 0.37 g/dL (IC 95% -0.02 to 0.75) increase in the hemoglobin concentration when compared to the children that were not infected at the beginning of the study. The children in whom the bacterium was eradicated and who received ferrous sulfate showed an increase of 0.47 g/dL (IC 95% 0.01 to 0.93) in hemoglobin concentration when compared with the students without infection who received ferrous sulfate. Students without H. pylori infection at the beginning of the study showed an increase in ferritin concentration of 11.3 ng/mL (IC 95% 1.9 a 20.6); students in whom the bacterium was
eliminated and received ferrous sulfate presented a 7.9 (IC 95% -2.2 to 18.1) increase when compared to the group were the infection was eradicated and received placebo. Conclusions: The results of these studies suggest that: H. pylori infection negatively affects the height of the students. The students with iron deficiency and/or anemia and H. pylori infection improve their iron nutritional status if they receive iron supplementation together with H. pylori eradication.
The Doctrine of 'Double Effect' and its Limitations Since the 13th century moral theologians have invoked the doctrine of 'double effect' to justify actions which result in both good and bad outcomes. It derives from the view that human life is a gift from God who alone may determine its span. It is used to support medical decisions designed to relieve suffering, where death becomes an uninte
IMMUNE FUNCTION IN ADULT HEART TRANSPLANT PATIENTS REFLECTS RISK FOR ORGAN REJECTION AND INFECTION Data from 76 patients over 3 years confirms value of assay of cell-mediated immunity Boston, MA, April 11, 2008 – Results of an analysis of three years of data on the monitoring of cell- mediated immunity (CMI) in adult patients undergoing heart transplantation at the University
35.9 and 14.5 per 106 PY for males and females, respectively. Specific municipalities were shown to have significantly higher rates, and may be appropriate for targeted screening programs.
Conclusion: We have found that use of a comprehensive endoscopy database in a well-defined