RESUMEN
BACKGROUND & AIMS: The amino acid hypusine, synthesized from the polyamine spermidine by the enzyme deoxyhypusine synthase (DHPS), is essential for the activity of eukaryotic translation initiation factor 5A (EIF5A). The role of hypusinated EIF5A (EIF5AHyp) remains unknown in intestinal homeostasis. Our aim was to investigate EIF5AHyp in the gut epithelium in inflammation and carcinogenesis. METHODS: We used human colon tissue messenger RNA samples and publicly available transcriptomic datasets, tissue microarrays, and patient-derived colon organoids. Mice with intestinal epithelial-specific deletion of Dhps were investigated at baseline and in models of colitis and colon carcinogenesis. RESULTS: We found that patients with ulcerative colitis and Crohn's disease exhibit reduced colon levels of DHPS messenger RNA and DHPS protein and reduced levels of EIF5AHyp. Similarly, colonic organoids from colitis patients also show down-regulated DHPS expression. Mice with intestinal epithelial-specific deletion of Dhps develop spontaneous colon hyperplasia, epithelial proliferation, crypt distortion, and inflammation. Furthermore, these mice are highly susceptible to experimental colitis and show exacerbated colon tumorigenesis when treated with a carcinogen. Transcriptomic and proteomic analysis on colonic epithelial cells demonstrated that loss of hypusination induces multiple pathways related to cancer and immune response. Moreover, we found that hypusination enhances translation of numerous enzymes involved in aldehyde detoxification, including glutathione S-transferases and aldehyde dehydrogenases. Accordingly, hypusination-deficient mice exhibit increased levels of aldehyde adducts in the colon, and their treatment with a scavenger of electrophiles reduces colitis. CONCLUSIONS: Hypusination in intestinal epithelial cells has a key role in the prevention of colitis and colorectal cancer, and enhancement of this pathway via supplementation of spermidine could have a therapeutic impact.
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Colitis , Espermidina , Humanos , Animales , Ratones , Espermidina/farmacología , Espermidina/metabolismo , Proteómica , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/metabolismo , Carcinogénesis/genética , Colitis/inducido químicamente , Colitis/genética , Colitis/prevención & control , Homeostasis , InflamaciónRESUMEN
Colonization by Helicobacter pylori is associated with gastric diseases, ranging from superficial gastritis to more severe pathologies, including intestinal metaplasia and adenocarcinoma. The interplay of the host response and the pathogen affect the outcome of disease. One major component of the mucosal response to H. pylori is the activation of a strong but inefficient immune response that fails to control the infection and frequently causes tissue damage. We have shown that polyamines can regulate H. pylori-induced inflammation. Chemical inhibition of ornithine decarboxylase (ODC), which generates the polyamine putrescine from l-ornithine, reduces gastritis in mice and adenocarcinoma incidence in gerbils infected with H. pylori However, we have also demonstrated that Odc deletion in myeloid cells enhances M1 macrophage activation and gastritis. Here we used a genetic approach to assess the specific role of gastric epithelial ODC during H. pylori infection. Specific deletion of the gene encoding for ODC in gastric epithelial cells reduces gastritis, attenuates epithelial proliferation, alters the metabolome, and downregulates the expression of immune mediators induced by H. pylori Inhibition of ODC activity or ODC knockdown in human gastric epithelial cells dampens H. pylori-induced NF-κB activation, CXCL8 mRNA expression, and IL-8 production. Chronic inflammation is a major risk factor for the progression to more severe pathologies associated with H. pylori infection, and we now show that epithelial ODC plays an important role in mediating this inflammatory response.
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Adenocarcinoma , Gastritis , Infecciones por Helicobacter , Helicobacter pylori , Adenocarcinoma/metabolismo , Animales , Células Epiteliales/metabolismo , Mucosa Gástrica/patología , Helicobacter pylori/metabolismo , Humanos , Inflamación/metabolismo , Ratones , Ornitina Descarboxilasa/genética , Ornitina Descarboxilasa/metabolismoRESUMEN
One of the major contributors to child mortality in the world is diarrheal diseases, with an estimated 800,000 deaths per year. Many pathogens are causative agents of these illnesses, including the enteropathogenic or enterohemorrhagic forms of Escherichia coli. These bacteria are characterized by their ability to cause attaching and effacing lesions in the gut mucosa. Although much has been learned about the pathogenicity of these organisms and the immune response against them, the role of the intestinal microbiota during these infections is not well characterized. Infection of mice with E. coli requires pre-treatment with antibiotics in most mouse models, which hinders the study of the microbiota in an undisturbed environment. Using Citrobacter rodentium as a murine model for attaching and effacing bacteria, we show that C57BL/6 mice deficient in granzyme B expression are highly susceptible to severe disease caused by C. rodentium infection. Although a previous publication from our group shows that granzyme B-deficient CD4+ T cells are partially responsible for this phenotype, in this report, we present data demonstrating that the microbiota, in particular members of the order Turicibacterales, have an important role in conferring resistance. Mice deficient in Turicibacter sanguinis have increased susceptibility to severe disease. However, when these mice are co-housed with resistant mice or colonized with T. sanguinis, susceptibility to severe infection is reduced. These results clearly suggest a critical role for this commensal in the protection against enteropathogens.
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Infecciones por Enterobacteriaceae , Escherichia coli , Niño , Humanos , Animales , Ratones , Citrobacter rodentium/genética , Granzimas , Infecciones por Enterobacteriaceae/microbiología , Ratones Endogámicos C57BL , BacteriasRESUMEN
BACKGROUND & AIMS: Because inflammatory bowel disease is increasing worldwide and can lead to colitis-associated carcinoma (CAC), new interventions are needed. We have shown that spermine oxidase (SMOX), which generates spermidine (Spd), regulates colitis. Here we determined whether Spd treatment reduces colitis and carcinogenesis. METHODS: SMOX was quantified in human colitis and associated dysplasia using quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. We used wild-type (WT) and Smox-/- C57BL/6 mice treated with dextran sulfate sodium (DSS) or azoxymethane (AOM)-DSS as models of colitis and CAC, respectively. Mice with epithelial-specific deletion of Apc were used as a model of sporadic colon cancer. Animals were supplemented or not with Spd in the drinking water. Colonic polyamines, inflammation, tumorigenesis, transcriptomes, and microbiomes were assessed. RESULTS: SMOX messenger RNA levels were decreased in human ulcerative colitis tissues and inversely correlated with disease activity, and SMOX protein was reduced in colitis-associated dysplasia. DSS colitis and AOM-DSS-induced dysplasia and tumorigenesis were worsened in Smox-/- vs WT mice and improved in both genotypes with Spd. Tumor development caused by Apc deletion was also reduced by Spd. Smox deletion and AOM-DSS treatment were both strongly associated with increased expression of α-defensins, which was reduced by Spd. A shift in the microbiome, with reduced abundance of Prevotella and increased Proteobacteria and Deferribacteres, occurred in Smox-/- mice and was reversed with Spd. CONCLUSIONS: Loss of SMOX is associated with exacerbated colitis and CAC, increased α-defensin expression, and dysbiosis of the microbiome. Spd supplementation reverses these phenotypes, indicating that it has potential as an adjunctive treatment for colitis and chemopreventive for colon carcinogenesis.
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Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Colitis/genética , Neoplasias del Colon/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Espermidina/uso terapéutico , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Azoximetano , Colitis/inducido químicamente , Colitis/enzimología , Colitis/prevención & control , Colitis Ulcerosa/enzimología , Colitis Ulcerosa/genética , Colon/enzimología , Colon/patología , Neoplasias del Colon/prevención & control , Sulfato de Dextran , Microbioma Gastrointestinal/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mucosa Intestinal/enzimología , Mucosa Intestinal/patología , Masculino , Ratones , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Lesiones Precancerosas/enzimología , Factores Protectores , ARN Mensajero/metabolismo , Índice de Severidad de la Enfermedad , Espermidina/metabolismo , Espermidina/farmacología , Pérdida de Peso/efectos de los fármacos , alfa-Defensinas/genética , alfa-Defensinas/metabolismo , Poliamino OxidasaRESUMEN
Innate immune responses are critical for mucosal immunity. Here we describe an innate lymphocyte population, iCD8α cells, characterized by expression of CD8α homodimers. iCD8α cells exhibit innate functional characteristics such as the capacity to engulf and kill bacteria. Development of iCD8α cells depends on expression of interleukin-2 receptor γ chain (IL-2Rγc), IL-15, and the major histocompatibility complex (MHC) class Ib protein H2-T3, also known as the thymus leukemia antigen or TL. While lineage tracking experiments indicated that iCD8α cells have a lymphoid origin, their development was independent of the transcriptional suppressor Id2, suggesting that these cells do not belong to the family of innate lymphoid cells. Finally, we identified cells with a similar phenotype in humans, which were profoundly depleted in newborns with necrotizing enterocolitis. These findings suggest a critical role of iCD8α cells in immune responses associated with the intestinal epithelium.
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Presentación de Antígeno/inmunología , Antígenos CD8/biosíntesis , Inmunidad Mucosa/inmunología , Mucosa Intestinal/citología , Linfocitos/inmunología , Animales , Citrobacter rodentium/inmunología , Citocalasina D/farmacología , Enterocolitis Necrotizante , Helicobacter pylori/inmunología , Antígenos de Histocompatibilidad Clase I/biosíntesis , Humanos , Proteína 2 Inhibidora de la Diferenciación/genética , Subunidad gamma Común de Receptores de Interleucina/biosíntesis , Interleucina-15/biosíntesis , Interleucina-2/biosíntesis , Interleucina-7/biosíntesis , Mucosa Intestinal/inmunología , Activación de Linfocitos/inmunología , Linfocitos/clasificación , Linfocitos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunologíaRESUMEN
BACKGROUND & AIMS: Helicobacter pylori eradication and endoscopic surveillance of gastric precancerous lesions are strategies to reduce gastric cancer (GC) risk. To our knowledge, this study is the longest prospective cohort of an H pylori eradication trial in a Hispanic population. METHODS: A total of 800 adults with precancerous lesions were randomized to anti-H pylori treatment or placebo. Gastric biopsy samples taken at baseline and 3, 6, 12, 16, and 20 years were assessed by our Correa histopathology score. A generalized linear mixed model with a participant-level random intercept was used to estimate the effect of H pylori status on the score over time. Logistic regression models were used to estimate progression by baseline diagnosis and to estimate GC risk by intestinal metaplasia (IM) subtype and anatomic location. RESULTS: Overall, 356 individuals completed 20 years of follow-up. Anti-H pylori therapy (intention-to-treat) reduced progression of the Correa score (odds ratio [OR], 0.59; 95% confidence interval [CI], 0.38-0.93). H pylori-negative status had a beneficial effect on the score over time (P = .036). Among individuals with IM (including indefinite for dysplasia) at baseline, incidence rates per 100 person-years were 1.09 (95% CI, 0.85-1.33) for low-grade/high-grade dysplasia and 0.14 (95% CI, 0.06-0.22) for GC. Incomplete-type (vs complete-type) IM at baseline presented higher GC risk (OR, 13.4; 95% CI, 1.8-103.8). Individuals with corpus (vs antrum-restricted) IM showed an OR of 2.1 (95% CI, 0.7-6.6) for GC. CONCLUSIONS: In a high-GC-risk Hispanic population, anti-H pylori therapy had a long-term beneficial effect against histologic progression. Incomplete IM is a strong predictor of GC risk.
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Antibacterianos/uso terapéutico , Mucosa Gástrica/patología , Infecciones por Helicobacter/tratamiento farmacológico , Lesiones Precancerosas/epidemiología , Neoplasias Gástricas/prevención & control , Adulto , Anciano , Biopsia , Colombia/epidemiología , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Mucosa Gástrica/diagnóstico por imagen , Mucosa Gástrica/microbiología , Gastroscopía/estadística & datos numéricos , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/aislamiento & purificación , Humanos , Incidencia , Masculino , Metaplasia/diagnóstico , Metaplasia/epidemiología , Metaplasia/microbiología , Metaplasia/patología , Persona de Mediana Edad , Lesiones Precancerosas/diagnóstico , Lesiones Precancerosas/microbiología , Lesiones Precancerosas/patología , Estudios Prospectivos , Factores de Riesgo , Neoplasias Gástricas/epidemiología , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/patología , Resultado del TratamientoRESUMEN
BACKGROUND & AIMS: Patients with inflammatory bowel disease (IBD) demonstrate nutritional selenium deficiencies and are at greater risk of developing colon cancer. Previously, we determined that global reduction of the secreted antioxidant selenium-containing protein, selenoprotein P (SELENOP), substantially increased tumor development in an experimental colitis-associated cancer (CAC) model. We next sought to delineate tissue-specific contributions of SELENOP to intestinal inflammatory carcinogenesis and define clinical context. METHODS: Selenop floxed mice crossed with Cre driver lines to delete Selenop from the liver, myeloid lineages, or intestinal epithelium were placed on an azoxymethane/dextran sodium sulfate experimental CAC protocol. SELENOP loss was assessed in human ulcerative colitis (UC) organoids, and expression was queried in human and adult UC samples. RESULTS: Although large sources of SELENOP, both liver- and myeloid-specific Selenop deletion failed to modify azoxymethane/dextran sodium sulfate-mediated tumorigenesis. Instead, epithelial-specific deletion increased CAC tumorigenesis, likely due to elevated oxidative stress with a resulting increase in genomic instability and augmented tumor initiation. SELENOP was down-regulated in UC colon biopsies and levels were inversely correlated with endoscopic disease severity and tissue S100A8 (calprotectin) gene expression. CONCLUSIONS: Although global selenium status is typically assessed by measuring liver-derived plasma SELENOP levels, our results indicate that the peripheral SELENOP pool is dispensable for CAC. Colonic epithelial SELENOP is the main contributor to local antioxidant capabilities. Thus, colonic SELENOP is the most informative means to assess selenium levels and activity in IBD patients and may serve as a novel biomarker for UC disease severity and identify patients most predisposed to CAC development.
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Colitis Ulcerosa/metabolismo , Neoplasias Asociadas a Colitis/prevención & control , Colitis/metabolismo , Colon/metabolismo , Mucosa Intestinal/metabolismo , Estrés Oxidativo , Selenoproteína P/metabolismo , Adolescente , Animales , Azoximetano , Estudios de Casos y Controles , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Niño , Preescolar , Colitis/inducido químicamente , Colitis/genética , Colitis Ulcerosa/genética , Neoplasias Asociadas a Colitis/inducido químicamente , Neoplasias Asociadas a Colitis/genética , Neoplasias Asociadas a Colitis/metabolismo , Colon/patología , Daño del ADN , Sulfato de Dextran , Modelos Animales de Enfermedad , Femenino , Inestabilidad Genómica , Humanos , Mucosa Intestinal/patología , Hígado/metabolismo , Masculino , Ratones Noqueados , Células Mieloides/metabolismo , Selenoproteína P/genéticaRESUMEN
BACKGROUND & AIMS: Inflammation in the gastrointestinal tract may lead to the development of cancer. Dicarbonyl electrophiles, such as isolevuglandins (isoLGs), are generated from lipid peroxidation during the inflammatory response and form covalent adducts with amine-containing macromolecules. Thus, we sought to determine the role of dicarbonyl electrophiles in inflammation-associated carcinogenesis. METHODS: The formation of isoLG adducts was analyzed in the gastric tissues of patients infected with Helicobacter pylori from gastritis to precancerous intestinal metaplasia, in human gastric organoids, and in patients with colitis and colitis-associated carcinoma (CAC). The effect on cancer development of a potent scavenger of dicarbonyl electrophiles, 5-ethyl-2-hydroxybenzylamine (EtHOBA), was determined in transgenic FVB/N insulin-gastrin (INS-GAS) mice and Mongolian gerbils as models of H pylori-induced carcinogenesis and in C57BL/6 mice treated with azoxymethane-dextran sulfate sodium as a model of CAC. The effect of EtHOBA on mutations in gastric epithelial cells of H pylori-infected INS-GAS mice was assessed by whole-exome sequencing. RESULTS: We show increased isoLG adducts in gastric epithelial cell nuclei in patients with gastritis and intestinal metaplasia and in human gastric organoids infected with H pylori. EtHOBA inhibited gastric carcinoma in infected INS-GAS mice and gerbils and attenuated isoLG adducts, DNA damage, and somatic mutation frequency. Additionally, isoLG adducts were elevated in tissues from patients with colitis, colitis-associated dysplasia, and CAC as well as in dysplastic tumors of C57BL/6 mice treated with azoxymethane-dextran sulfate sodium. In this model, EtHOBA significantly reduced adduct formation, tumorigenesis, and dysplasia severity. CONCLUSIONS: Dicarbonyl electrophiles represent a link between inflammation and somatic genomic alterations and are thus key targets for cancer chemoprevention.
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Transformación Celular Neoplásica/inmunología , Neoplasias Asociadas a Colitis/inmunología , Lípidos/inmunología , Lesiones Precancerosas/inmunología , Neoplasias Gástricas/inmunología , Animales , Bencilaminas/farmacología , Bencilaminas/uso terapéutico , Núcleo Celular/metabolismo , Transformación Celular Neoplásica/efectos de los fármacos , Neoplasias Asociadas a Colitis/microbiología , Neoplasias Asociadas a Colitis/patología , Neoplasias Asociadas a Colitis/prevención & control , Modelos Animales de Enfermedad , Células Epiteliales , Mucosa Gástrica/citología , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/inmunología , Mucosa Gástrica/patología , Gastritis/inmunología , Gastritis/microbiología , Gastritis/patología , Gerbillinae , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/inmunología , Helicobacter pylori/aislamiento & purificación , Humanos , Lípidos/antagonistas & inhibidores , Metaplasia/inmunología , Metaplasia/microbiología , Metaplasia/patología , Ratones , Ratones Transgénicos , Organoides , Lesiones Precancerosas/tratamiento farmacológico , Lesiones Precancerosas/microbiología , Lesiones Precancerosas/patología , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/patología , Neoplasias Gástricas/prevención & controlRESUMEN
Infection by Helicobacter pylori is the primary cause of gastric adenocarcinoma. The most potent H. pylori virulence factor is cytotoxin-associated gene A (CagA), which is translocated by a type 4 secretion system (T4SS) into gastric epithelial cells and activates oncogenic signaling pathways. The gene cagY encodes for a key component of the T4SS and can undergo gene rearrangements. We have shown that the cancer chemopreventive agent α-difluoromethylornithine (DFMO), known to inhibit the enzyme ornithine decarboxylase, reduces H. pylori-mediated gastric cancer incidence in Mongolian gerbils. In the present study, we questioned whether DFMO might directly affect H. pylori pathogenicity. We show that H. pylori output strains isolated from gerbils treated with DFMO exhibit reduced ability to translocate CagA in gastric epithelial cells. Further, we frequently detected genomic modifications in the middle repeat region of the cagY gene of output strains from DFMO-treated animals, which were associated with alterations in the CagY protein. Gerbils did not develop carcinoma when infected with a DFMO output strain containing rearranged cagY or the parental strain in which the wild-type cagY was replaced by cagY with DFMO-induced rearrangements. Lastly, we demonstrate that in vitro treatment of H. pylori by DFMO induces oxidative DNA damage, expression of the DNA repair enzyme MutS2, and mutations in cagY, demonstrating that DFMO directly affects genomic stability. Deletion of mutS2 abrogated the ability of DFMO to induce cagY rearrangements directly. In conclusion, DFMO-induced oxidative stress in H. pylori leads to genomic alterations and attenuates virulence.
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Proteínas Bacterianas/genética , Carcinogénesis/genética , Carcinogénesis/patología , Eflornitina/farmacología , Helicobacter pylori/genética , Mutación/genética , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/patología , Animales , Daño del ADN , Eliminación de Gen , Reordenamiento Génico , Gerbillinae , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/patogenicidad , Masculino , Estrés Oxidativo/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , VirulenciaRESUMEN
BACKGROUND & AIMS: Countries endemic for parasitic infestations have a lower incidence of Crohn's disease (CD) than nonendemic countries, and there have been anecdotal reports of the beneficial effects of helminths in CD patients. Tuft cells in the small intestine sense and direct the immune response against eukaryotic parasites. We investigated the activities of tuft cells in patients with CD and mouse models of intestinal inflammation. METHODS: We used microscopy to quantify tuft cells in intestinal specimens from patients with ileal CD (n = 19), healthy individuals (n = 14), and TNFΔARE/+ mice, which develop Crohn's-like ileitis. We performed single-cell RNA sequencing, mass spectrometry, and microbiome profiling of intestinal tissues from wild-type and Atoh1-knockout mice, which have expansion of tuft cells, to study interactions between microbes and tuft cell populations. We assessed microbe dependence of tuft cell populations using microbiome depletion, organoids, and microbe transplant experiments. We used multiplex imaging and cytokine assays to assess alterations in inflammatory response following expansion of tuft cells with succinate administration in TNFΔARE/+ and anti-CD3E CD mouse models. RESULTS: Inflamed ileal tissues from patients and mice had reduced numbers of tuft cells, compared with healthy individuals or wild-type mice. Expansion of tuft cells was associated with increased expression of genes that regulate the tricarboxylic acid cycle, which resulted from microbe production of the metabolite succinate. Experiments in which we manipulated the intestinal microbiota of mice revealed the existence of an ATOH1-independent population of tuft cells that was sensitive to metabolites produced by microbes. Administration of succinate to mice expanded tuft cells and reduced intestinal inflammation in TNFΔARE/+ mice and anti-CD3E-treated mice, increased GATA3+ cells and type 2 cytokines (IL22, IL25, IL13), and decreased RORGT+ cells and type 17 cytokines (IL23) in a tuft cell-dependent manner. CONCLUSIONS: We found that tuft cell expansion reduced chronic intestinal inflammation in mice. Strategies to expand tuft cells might be developed for treatment of CD.
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Células Quimiorreceptoras/inmunología , Enfermedad de Crohn/inmunología , Microbioma Gastrointestinal/inmunología , Ileítis/inmunología , Mucosa Intestinal/inmunología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Células Quimiorreceptoras/patología , Enfermedad de Crohn/microbiología , Enfermedad de Crohn/patología , ADN Bacteriano/genética , Modelos Animales de Enfermedad , Heces/microbiología , Femenino , Humanos , Ileítis/microbiología , Ileítis/patología , Íleon/citología , Íleon/inmunología , Íleon/microbiología , Íleon/patología , Mucosa Intestinal/citología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Noqueados , Factores Protectores , ARN Ribosómico 16S/genética , RNA-Seq , Análisis de la Célula Individual , Ácido Succínico/inmunología , Ácido Succínico/metabolismoRESUMEN
Colombia, South America has one of the world's highest burdens of Helicobacter pylori infection and gastric cancer. While multidrug antibiotic regimens can effectively eradicate H. pylori, treatment efficacy is being jeopardized by the emergence of antibiotic-resistant H. pylori strains. Moreover, the spectrum of and genetic mechanisms for antibiotic resistance in Colombia is underreported. In this study, 28 H. pylori strains isolated from gastric biopsy specimens from a high-gastric-cancer-risk (HGCR) population living in the Andes Mountains in Túquerres, Colombia and 31 strains from a low-gastric-cancer-risk (LGCR) population residing on the Pacific coast in Tumaco, Colombia were subjected to antibiotic susceptibility testing for amoxicillin, clarithromycin, levofloxacin, metronidazole, rifampin, and tetracycline. Resistance-associated genes were amplified by PCR for all isolates, and 29 isolates were whole-genome sequenced (WGS). No strains were resistant to amoxicillin, clarithromycin, or rifampin. One strain was resistant to tetracycline and had an A926G mutation in its 16S rRNA gene. Levofloxacin resistance was observed in 12/59 isolates and was significantly associated with N87I/K and/or D91G/Y mutations in gyrA Most isolates were resistant to metronidazole; this resistance was significantly higher in the LGCR (31/31) group compared to the HGCR (24/28) group. Truncations in rdxA and frxA were present in nearly all metronidazole-resistant strains. There was no association between phylogenetic relationship and resistance profiles based on WGS analysis. Our results indicate H. pylori isolates from Colombians exhibit multidrug antibiotic resistance. Continued surveillance of H. pylori antibiotic resistance in Colombia is warranted in order to establish appropriate eradication treatment regimens for this population.
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Infecciones por Helicobacter , Helicobacter pylori , Neoplasias Gástricas , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Claritromicina/farmacología , Colombia/epidemiología , Farmacorresistencia Bacteriana/genética , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/epidemiología , Helicobacter pylori/genética , Humanos , Metronidazol/farmacología , Pruebas de Sensibilidad Microbiana , Filogenia , ARN Ribosómico 16S/genética , ARN Ribosómico 23S , América del Sur , Neoplasias Gástricas/tratamiento farmacológicoRESUMEN
Helicobacter pylori is the strongest risk factor for gastric cancer. Initial interactions between H. pylori and its host originate at the microbial-gastric epithelial cell interface, and contact between H. pylori and gastric epithelium activates signaling pathways that drive oncogenesis. One microbial constituent that increases gastric cancer risk is the cag pathogenicity island, which encodes a type IV secretion system that translocates the effector protein, CagA, into host cells. We previously demonstrated that infection of Mongolian gerbils with a carcinogenic cag+H. pylori strain, 7.13, recapitulates many features of H. pylori-induced gastric cancer in humans. Therefore, we sought to define gastric proteomic changes induced by H. pylori that are critical for initiation of the gastric carcinogenic cascade. Gastric cell scrapings were harvested from H. pylori-infected and uninfected gerbils for quantitative proteomic analyses using isobaric tags for relative and absolute quantitation (iTRAQ). Quantitative proteomic analysis of samples from two biological replicate experiments quantified a total of 2764 proteins, 166 of which were significantly altered in abundance by H. pylori infection. Pathway mapping identified significantly altered inflammatory and cancer-signaling pathways that included Rab/Ras signaling proteins. Consistent with the iTRAQ results, RABEP2 and G3BP2 were significantly up-regulated in vitro, ex vivo in primary human gastric monolayers, and in vivo in gerbil gastric epithelium following infection with H. pylori strain 7.13 in a cag-dependent manner. Within human stomachs, RABEP2 and G3BP2 expression in gastric epithelium increased in parallel with the severity of premalignant and malignant lesions and was significantly elevated in intestinal metaplasia and dysplasia, as well as gastric adenocarcinoma, compared with gastritis alone. These results indicate that carcinogenic strains of H. pylori induce dramatic and specific changes within the gastric proteome in vivo and that a subset of altered proteins within pathways with oncogenic potential may facilitate the progression of gastric carcinogenesis in humans.
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Proteínas Portadoras/metabolismo , Infecciones por Helicobacter/complicaciones , Helicobacter pylori/patogenicidad , Neoplasias Gástricas/microbiología , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Línea Celular , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Gerbillinae , Infecciones por Helicobacter/microbiología , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Masculino , Mapas de Interacción de Proteínas , Proteómica , Proteínas de Unión al ARN , Neoplasias Gástricas/metabolismo , Regulación hacia ArribaRESUMEN
Helicobacter pylori colonizes the gastric mucosa and secretes a pore-forming toxin (VacA). Two main types of VacA, m1 and m2, can be distinguished by phylogenetic analysis. Type m1 forms of VacA have been extensively studied, but there has been relatively little study of m2 forms. In this study, we generated H. pylori strains producing chimeric proteins in which VacA m1 segments of a parental strain were replaced by corresponding m2 sequences. In comparison to the parental m1 VacA protein, a chimeric protein (designated m2/m1) containing m2 sequences in the N-terminal portion of the m region was less potent in causing vacuolation of HeLa cells, AGS gastric cells, and AZ-521 duodenal cells and had reduced capacity to cause membrane depolarization or death of AZ-521 cells. Consistent with the observed differences in activity, the chimeric m2/m1 VacA protein bound to cells at reduced levels compared to the binding levels of the parental m1 protein. The presence of two strain-specific insertions or deletions within or adjacent to the m region did not influence toxin activity. Experiments with human gastric organoids grown as monolayers indicated that m1 and m2/m1 forms of VacA had similar cell-vacuolating activities. Interestingly, both forms of VacA bound preferentially to the basolateral surface of organoid monolayers and caused increased cell vacuolation when interacting with the basolateral surface compared to the apical surface. These data provide insights into functional correlates of sequence variation in the VacA midregion (m region).
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Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Variación Genética , Infecciones por Helicobacter/microbiología , Helicobacter pylori/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Humanos , Dominios Proteicos , Multimerización de Proteína , Transporte de Proteínas , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
The connection between inflammation and cancer was initially recognized by Rudolf Virchow in the nineteenth century. During the last decades, a large body of evidence has provided support to his hypothesis, and now inflammation is recognized as one of the hallmarks of cancer, both in etiopathogenesis and ongoing tumor growth. Infection with the pathogen Helicobacter pylori is the primary causal factor in 90% of gastric cancer (GC) cases. As we increase our understanding of how chronic inflammation develops in the stomach and contributes to carcinogenesis, there is increasing interest in targeting cancer-promoting inflammation as a strategy to treat GC. Moreover, once cancer develops and anti-cancer immune responses are suppressed, there is evidence of a substantial shift in the microenvironment and new targets for immune therapy emerge. In this chapter, we provide insight into inflammation-related factors, including T lymphocytes, macrophages, pro-inflammatory chemokines, and cytokines, which promote H. pylori-associated GC initiation and growth. While intervening with chronic inflammation is not a new practice in rheumatology or gastroenterology, this approach has not been fully explored for its potential to prevent carcinogenesis or to contribute to the treatment of GC. This review highlights current and possible strategies for therapeutic intervention including (i) targeting pro-inflammatory mediators, (ii) targeting growth factors and pathways involved in angiogenesis in the gastric tumor microenvironment, and (iii) enhancing anti-tumor immunity. In addition, we highlight a significant number of clinical trials and discuss the importance of individual tumor characterization toward offering personalized immune-related therapy.
Asunto(s)
Inflamación/inmunología , Inflamación/terapia , Neoplasias Gástricas/patología , Neoplasias Gástricas/terapia , Citocinas/inmunología , Mucosa Gástrica/inmunología , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Infecciones por Helicobacter/terapia , Helicobacter pylori/patogenicidad , Humanos , Inflamación/microbiología , Inflamación/patología , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/microbiología , Microambiente TumoralRESUMEN
Naturally occurring polyamines are ubiquitously distributed and play important roles in cell development, amino acid and protein synthesis, oxidative DNA damage, proliferation, and cellular differentiation. Macrophages are essential in the innate immune response, and contribute to tissue remodeling. Naïve macrophages have two major potential fates: polarization to (1) the classical pro-inflammatory M1 defense response to bacterial pathogens and tumor cells, and (2) the alternatively activated M2 response, induced in the presence of parasites and wounding, and also implicated in the development of tumor-associated macrophages. ODC, the rate-limiting enzyme in polyamine synthesis, leads to an increase in putrescine levels, which impairs M1 gene transcription. Additionally, spermidine and spermine can regulate translation of pro-inflammatory mediators in activated macrophages. In this review, we focus on polyamines in macrophage activation patterns in the context of gastrointestinal inflammation and carcinogenesis. We seek to clarify mechanisms of innate immune regulation by polyamine metabolism and potential novel therapeutic targets.
Asunto(s)
Macrófagos/inmunología , Poliaminas/inmunología , Animales , Polaridad Celular , Humanos , Activación de Macrófagos , Macrófagos/citología , Transcripción GenéticaRESUMEN
Macrophage activation is a critical step in host responses during bacterial infections. Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine metabolism, has been well studied in epithelial cells and is known to have essential roles in many different cellular functions. However, its role in regulating macrophage function during bacterial infections is not well characterized. We demonstrate that macrophage-derived ODC is a critical regulator of M1 macrophage activation during both Helicobacter pylori and Citrobacter rodentium infection. Myeloid-specific Odc deletion significantly increased gastric and colonic inflammation, respectively, and enhanced M1 activation. Add-back of putrescine, the product of ODC, reversed the increased macrophage activation, indicating that ODC and putrescine are regulators of macrophage function. Odc-deficient macrophages had increased histone 3, lysine 4 (H3K4) monomethylation, and H3K9 acetylation, accompanied by decreased H3K9 di/trimethylation both in vivo and ex vivo in primary macrophages. These alterations in chromatin structure directly resulted in up-regulated gene transcription, especially M1 gene expression. Thus, ODC in macrophages tempers antimicrobial, M1 macrophage responses during bacterial infections through histone modifications and altered euchromatin formation, leading to the persistence and pathogenesis of these organisms.
Asunto(s)
Infecciones por Enterobacteriaceae/inmunología , Infecciones por Helicobacter/inmunología , Histonas/metabolismo , Macrófagos/inmunología , Ornitina Descarboxilasa/inmunología , Animales , Línea Celular , Citrobacter rodentium , Colitis/inmunología , Colitis/patología , Colon/inmunología , Colon/patología , Citocinas/inmunología , Infecciones por Enterobacteriaceae/patología , Mucosa Gástrica/inmunología , Mucosa Gástrica/patología , Gastritis/inmunología , Gastritis/patología , Infecciones por Helicobacter/patología , Helicobacter pylori , Humanos , Activación de Macrófagos , Masculino , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Ornitina Descarboxilasa/genética , Putrescina/metabolismoRESUMEN
BACKGROUND: Geospatial technology has facilitated the discovery of disease distributions and etiology and helped target prevention programs. Globally, gastric cancer is the leading infection-associated cancer, and third leading cause of cancer mortality worldwide, with marked geographic variation. Central and South America have a significant burden, particularly in the mountainous regions. In the context of an ongoing population-based case-control study in Central America, our aim was to examine the spatial epidemiology of gastric cancer subtypes and H. pylori virulence factors. METHODS: Patients diagnosed with gastric cancer from 2002 to 2013 in western Honduras were identified in the prospective gastric cancer registry at the principal district hospital. Diagnosis was based on endoscopy and confirmatory histopathology. Geospatial methods were applied using the ArcGIS v10.3.1 and SaTScan v9.4.2 platforms to examine regional distributions of the gastric cancer histologic subtypes (Lauren classification), and the H. pylori CagA virulence factor. Getis-Ord-Gi hot spot and Discrete Poisson SaTScan statistics, respectively, were used to explore spatial clustering at the village level (30-50 rural households), with standardization by each village's population. H. pylori and CagA serologic status was determined using the novel H. pylori multiplex assay (DKFZ, Germany). RESULTS: Three hundred seventy-eight incident cases met the inclusion criteria (mean age 63.7, male 66.3%). Areas of higher gastric cancer incidence were identified. Significant spatial clustering of diffuse histology adenocarcinoma was revealed both by the Getis-Ord-GI* hot spot analysis (P-value < 0.0015; range 0.00003-0.0014; 99%CI), and by the SaTScan statistic (P-value < 0.006; range 0.0026-0.0054). The intestinal subtype was randomly distributed. H. pylori CagA had significant spatial clustering only in association with the diffuse histology cancer hot spot (Getis-Ord-Gi* P value ≤0.001; range 0.0001-0.0010; SaTScan statistic P value 0.0085). In the diffuse gastric cancer hot spot, the lowest age quartile range was 21-46 years, significantly lower than the intestinal cancers (P = 0.024). CONCLUSIONS: Geospatial methods have identified a significant cluster of incident diffuse type adenocarcinoma cases in rural Central America, suggest of a germline genetic association. Further genomic and geospatial analyses to identify potential spatial patterns of genetic, bacterial, and environmental risk factors may be informative.
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Salud Rural , Neoplasias Gástricas/epidemiología , Anciano , Estudios de Casos y Controles , América Central/epidemiología , Susceptibilidad a Enfermedades , Femenino , Geografía , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/microbiología , Helicobacter pylori , Humanos , Masculino , Persona de Mediana Edad , Medición de Riesgo , Factores de Riesgo , Análisis Espacial , Neoplasias Gástricas/etiología , Neoplasias Gástricas/patologíaRESUMEN
BACKGROUND: Antimicrobial resistance is a global public health problem, particularly in low- and middle-income countries (LMICs), where antibiotics are often obtained without a prescription. H. pylori antimicrobial resistance patterns are informative for patient care and gastric cancer prevention programs, have been shown to correlate with general antimicrobial consumption, and may guide antimicrobial stewardship programs in LMICs. We report H. pylori resistance and antimicrobial utilization patterns for western Honduras, representative of rural Central America. METHODS: In the context of the western Honduras gastric cancer epidemiology initiative, gastric biopsies from 189 patients were studied for culture and resistance patterns. Antimicrobial utilization was investigated for common H. pylori treatment regimens from regional public (7 antimicrobials) and national private (4 antimicrobials) data, analyzed in accordance with WHO anatomical therapeutic chemical defined daily doses (DDD) method and expressed as DDD/1000 inhabitants per day (DID) and per year (DIY). RESULTS: H. pylori was successfully cultured from 116 patients (56% males, mean age: 54), and nearly all strains were cagA+ and vacAs1m1+ positive (99% and 90.4%, respectively). Unexpectedly, high resistance was noted for levofloxacin (20.9%) and amoxicillin (10.7%), while metronidazole (67.9%) and clarithromycin (11.2%) were similar to data from Latin America. Significant associations with age, gender, or histology were not noted, with the exception of levofloxacin (28%, P = 0.01) in those with histology limited to non-atrophic gastritis. Total antimicrobial usage in western Honduras of amoxicillin (17.3 DID) and the quinolones had the highest relative utilizations compared with other representative nations. CONCLUSIONS: We observed significant H. pylori resistance to amoxicillin and levofloxacin in the context of high community antimicrobial utilization. This has implications in Central America for H. pylori treatment guidelines as well as antimicrobial stewardship programs.
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Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana , Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori/efectos de los fármacos , Adulto , Anciano , Amoxicilina/uso terapéutico , América Central , Femenino , Infecciones por Helicobacter/microbiología , Helicobacter pylori/clasificación , Helicobacter pylori/genética , Helicobacter pylori/aislamiento & purificación , Humanos , Levofloxacino/uso terapéutico , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana EdadRESUMEN
OBJECTIVE: Gastric cancer is the third leading cause of cancer death worldwide and infection by Helicobacter pylori is the strongest risk factor. We have reported increased epidermal growth factor receptor (EGFR) phosphorylation in the H. pylori-induced human carcinogenesis cascade, and association with DNA damage. Our goal was to determine the role of EGFR activation in gastric carcinogenesis. DESIGN: We evaluated gefitinib, a specific EGFR inhibitor, in chemoprevention of H. pylori-induced gastric inflammation and cancer development. Mice with genetically targeted epithelial cell-specific deletion of Egfr (EfgrΔepi mice) were also used. RESULTS: In C57BL/6 mice, gefitinib decreased Cxcl1 and Cxcl2 expression by gastric epithelial cells, myeloperoxidase-positive inflammatory cells in the mucosa and epithelial DNA damage induced by H. pylori infection. Similar reductions in chemokines, inflammatory cells and DNA damage occurred in infected EgfrΔepi versus Egfrfl/fl control mice. In H. pylori-infected transgenic insulin-gastrin (INS-GAS) mice and gerbils, gefitinib treatment markedly reduced dysplasia and carcinoma. Gefitinib blocked H. pylori-induced activation of mitogen-activated protein kinase 1/3 (MAPK1/3) and activator protein 1 in gastric epithelial cells, resulting in inhibition of chemokine synthesis. MAPK1/3 phosphorylation and JUN activation was reduced in gastric tissues from infected wild-type and INS-GAS mice treated with gefitinib and in primary epithelial cells from EfgrΔepi versus Egfrfl/fl mice. Epithelial EGFR activation persisted in humans and mice after H. pylori eradication, and gefitinib reduced gastric carcinoma in INS-GAS mice treated with antibiotics. CONCLUSIONS: These findings suggest that epithelial EGFR inhibition represents a potential strategy to prevent development of gastric carcinoma in H. pylori-infected individuals.
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Antineoplásicos/uso terapéutico , Receptores ErbB/antagonistas & inhibidores , Gastritis/patología , Infecciones por Helicobacter/patología , Quinazolinas/uso terapéutico , Neoplasias Gástricas/prevención & control , Animales , Técnicas de Cultivo de Célula , Células Epiteliales , Gastritis/microbiología , Gefitinib , Gerbillinae , Helicobacter pylori , Ratones , Ratones Endogámicos C57BL , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/patologíaRESUMEN
OBJECTIVE: Helicobacter pylori is the strongest risk factor for gastric cancer; however, the majority of infected individuals do not develop disease. Pathological outcomes are mediated by complex interactions among bacterial, host and environmental constituents, and two dietary factors linked with gastric cancer risk are iron deficiency and high salt. We hypothesised that prolonged adaptation of H. pylori to in vivo carcinogenic microenvironments results in genetic modification important for disease. DESIGN: Whole genome sequencing of genetically related H. pylori strains that differ in virulence and targeted H. pylori sequencing following prolonged exposure of bacteria to in vitro carcinogenic conditions were performed. RESULTS: A total of 180 unique single nucleotide polymorphisms (SNPs) were identified among the collective genomes when compared with a reference H. pylori genome. Importantly, common SNPs were identified in isolates harvested from iron-depleted and high salt carcinogenic microenvironments, including an SNP within fur (FurR88H). To investigate the direct role of low iron and/or high salt, H. pylori was continuously cultured in vitro under low iron or high salt conditions to assess fur genetic variation. Exposure to low iron or high salt selected for the FurR88H variant after only 5 days. To extend these results, fur was sequenced in 339 clinical H. pylori strains. Among the isolates examined, 17% (40/232) of strains isolated from patients with premalignant lesions harboured the FurR88H variant, compared with only 6% (6/107) of strains from patients with non-atrophic gastritis alone (p=0.0034). CONCLUSION: These results indicate that specific genetic variation arises within H. pylori strains during in vivo adaptation to conditions conducive for gastric carcinogenesis.