MNPmApp: An image analysis tool to quantify mononuclear phagocyte distribution in mucosal tissues.

Mononuclear phagocytes (MNPs) such as dendritic cells and macrophages perform key sentinel functions in mucosal tissues and are responsible for inducing and maintaining adaptive immune responses to mucosal pathogens. Positioning of MNPs at the epithelial interface facilitates their access to luminally-derived antigens and regulates MNP function through soluble mediators or surface receptor interactions. Therefore, accurately quantifying the distribution of MNPs within mucosal tissues as well as their spatial relationship with other cells is important to infer functional cellular interactions in health and disease. In this study, we developed and validated a MATLAB-based tissue cytometry platform, termed "MNP mapping application" (MNPmApp), that performs high throughput analyses of MNP density and distribution in the gastrointestinal mucosa based on digital multicolor fluorescence microscopy images and that integrates a Monte Carlo modeling feature to assess randomness of MNP distribution. MNPmApp identified MNPs in tissue sections of the human gastric mucosa with 98 ± 2% specificity and 76 ± 15% sensitivity for HLA-DR+ MNPs and 98 ± 1% specificity and 85 ± 12% sensitivity for CD11c+ MNPs. Monte Carlo modeling revealed that mean MNP-MNP distances for both HLA-DR+ and CD11c+ MNPs were significantly lower than anticipated based on random cell placement, whereas MNP-epithelial distances were similar to randomly placed cells. Surprisingly, H. pylori infection had no significant impact on the number of HLA-DR and CD11c MNPs or their distribution within the gastric lamina propria. However, our study demonstrated that MNPmApp is a reliable and user-friendly tool for unbiased quantitation of MNPs and their distribution at mucosal sites.

Helicobacter pylori infection and UBT-13C values are associated with changes in body mass index in children and adults.

BACKGROUND: The urea breath test (UBT-13C) is a non-invasive technique that allows the diagnosis and confirmation of eradication of Helicobacter pylori infection. AIM: To evaluate H. pylori positivity and values of UBT-13C among infected Chilean children and adults, and to analyze its variation in relation to sex, nutritional status, and age of the patients. MATERIAL AND METHODS: Retrospective study of 1141 patients aged 6 to 94 years, with an indication for a UBT-13C either for diagnosis or for confirmation of eradication of H. pylori infection. 13C enrichment was measured using an infrared spectrometer calculating the delta 13C values before and after the ingestion of 13C marked urea. The clinical data of the patients were obtained at the time of the examination. RESULTS: We included 241 children and 900 adults. Infected children obtained lower UBT-13C delta values than infected adults (16.1 ± 8.7 and 37 ± 52.9, respectively). The rates of infection were higher in males who were recruited for diagnosis. Significant differences were obtained between positivity for H. pylori in overweight and obese children but not adults. UBT-13C titers were significantly associated with the body mass index (BMI) only in adults. CONCLUSIONS: H. pylori infection rates are similar between sexes and are higher in children probably because of selection bias. In children, H. pylori positivity is associated with higher BMI and excess malnutrition although with similar UBT-13C values. In adults, H. pylori infection is not related with BMI, but a higher BMI impacts UBT-13C titers.

High Helicobacter pylori Bacterial Load and Low Cytokine Expression Levels Are Associated with Nodular Gastropathy.

BACKGROUND AND AIMS: Nodular gastropathy (NG) is an inflammatory condition of the gastric mucosa characterized by the endoscopic detection of multiple millimeter protrusions. A strong association between NG and Helicobacter pylori and a possible role of NG as a risk factor for undifferentiated gastric cancer have been described. The aim of this study was to characterize the pathogenic and inflammatory profile of patients with NG. METHODS: Adult patients referred for upper gastrointestinal endoscopy were prospectively enrolled in this study. H. pylori infection status was determined by rapid urease test. Biopsies were stained with hematoxylin-eosin. Sydney and OLGA scores were used to assess gastritis characteristics and gastric cancer risk. PCR analysis was performed to determine bacterial load and virulence factors CagA (and its EPIYA motifs) and VacA alleles. Finally, gastric mucosa cytokine gene expression (IL-8, IL-1ß, and TNF-α) was determined by real-time RT-PCR. RESULTS: Forty-eight patients, mean age of 36 years, were recruited. All NG patients were infected by H. pylori. OLGA score was similar in both groups (NG patients and non-NG patients). NG patients had higher bacterial load in the gastric corpus (p = 0.01) and significantly less pro-inflammatory cytokine levels than non-NG infected patients (p = 0.01). CONCLUSIONS: In our study, NG is not associated with preneoplastic lesions. An increase in bacterial load without a concomitant increase in mucosal inflammatory cytokine responses in H. pylori-infected subjects with NG may represent a general dampening of immune responses or an additional mechanism of H. pylori active immune evasion.

Early origins of allergy and asthma (ARIES): study protocol for a prospective prenatal birth cohort in Chile.

BACKGROUND: Growing evidence shows that atopic dermatitis (AD), food allergy (FA), allergic rhinitis, and asthma are largely determined during the first 1000 days (time elapsed from conception to the 2nd birthday). The ARIES birth cohort aims to determine prenatal and perinatal conditions, as well as genetic and epigenetic factors, that participate in the early setting of immune responses, and the role of these in the later determination of the risk of allergic diseases and asthma in the offspring. METHODS: We have designed a birth cohort of 250 families with prenatal recruitment (~ 14 weeks). We will genotype relevant allergy/asthma-associated variants in trios and will perform immunophenotyping and evaluation of allergy biomarkers in cord blood. At 1 and 2 years of age we will assess if infants have developed allergic sensitization, AD, FA, as well as biomarkers of asthma including the asthma predictive index. We will also evaluate how maternal conditions modify immune programming through epigenetic modifications and will then depict newborn epigenetic cues of allergy/asthma risk. Next, we will assess composition/diversity of maternal gut, placenta, breastmilk and infant gut microbiome and their association with immunophenotype and biomarkers at birth, and clinical outcomes at age 1 and 2. Finally, we plan to assess how environmental exposures (perinatal outdoor and indoor pollution, allergens and endotoxin) affect the incidence of allergic sensitization, AD, FA, and risk of asthma. DISCUSSION: The in-depth study of the ARIES birth cohort shall provide crucial information to understand the rising incidence of allergies and asthma in developing countries, and hopefully provide cues on how to prevent and treat these diseases. TRIAL REGISTRATION: clinicaltrials.gov NCT04186949, retrospectively registered on December 5, 2019.

[Adaptation to the reality of Latin America of the NASPGHAN/ESPGHAN 2016 Guidelines on the Diagnosis, Prevention and Treatment of Helicobacter pylori Infection in Pediatrics]. / Adaptación a la realidad de Latinoamérica de la Guía Clínica NASPGHAN/ESPGHAN 2016 sobre Diagnóstico, Prevención y Tratamiento de Infección por Helicobacter pylori en Pediatría.

INTRODUCTION: The latest joint H. pylori NASPGHAN and ESPGHAN clinical guidelines published in 2016, contain 20 statements that have been questioned in practice regarding their applicability in Latin America (LA); in particular in relation to gastric cancer prevention. METHODS: We conduc ted a critical analysis of the literature, with special emphasis on LA data and established the level of evidence and level of recommendation of the most controversial claims in the Joint Guidelines. Two rounds of voting were conducted according to the Delphi consensus technique and a Likert scale (from 0 to 4) was used to establish the "degree of agreement" among a panel of SLAGHNP ex perts. RESULTS: There are few studies regarding diagnosis, treatment effectiveness and susceptibility to antibiotics of H. pylori in pediatric patients of LA. Based on these studies, extrapolations from adult studies, and the clinical experience of the participating expert panel, the following recom mendations are made. We recommend taking biopsies for rapid urease and histology testing (and samples for culture or molecular techniques, when available) during upper endoscopy only if in case of confirmed H. pylori infection, eradication treatment will be indicated. We recommend that selected regional centers conduct antimicrobial sensitivity/resistance studies for H. pylori and thus act as reference centers for all LA. In case of failure to eradicate H. pylori with first-line treatment, we recommend empirical treatment with quadruple therapy with proton pump inhibitor, amoxi cillin, metronidazole, and bismuth for 14 days. In case of eradication failure with the second line scheme, it is recommended to indicate an individualized treatment considering the age of the pa tient, the previously indicated scheme and the antibiotic sensitivity of the strain, which implies performing a new endoscopy with sample extraction for culture and antibiogram or molecular resistance study. In symptomatic children referred to endoscopy who have a history of first or se cond degree family members with gastric cancer, it is recommended to consider the search for H. pylori by direct technique during endoscopy (and eradicate it when detected). CONCLUSIONS: The evidence supports most of the general concepts of the NASPGHAN/ESPGHAN 2016 Guidelines, but it is necessary to adapt them to the reality of LA, with emphasis on the development of regional centers for the study of antibiotic sensitivity and to improve the correct selection of the eradication treatment. In symptomatic children with a family history of first or second degree gastric cancer, the search for and eradication of H. pylori should be considered.

Regulation of Rac1 and Reactive Oxygen Species Production in Response to Infection of Gastrointestinal Epithelia.

Generation of reactive oxygen species (ROS) during infection is an immediate host defense leading to microbial killing. APE1 is a multifunctional protein induced by ROS and after induction, protects against ROS-mediated DNA damage. Rac1 and NAPDH oxidase (Nox1) are important contributors of ROS generation following infection and associated with gastrointestinal epithelial injury. The purpose of this study was to determine if APE1 regulates the function of Rac1 and Nox1 during oxidative stress. Gastric or colonic epithelial cells (wild-type or with suppressed APE1) were infected with Helicobacter pylori or Salmonella enterica and assessed for Rac1 and NADPH oxidase-dependent superoxide production. Rac1 and APE1 interactions were measured by co-immunoprecipitation, confocal microscopy and proximity ligation assay (PLA) in cell lines or in biopsy specimens. Significantly greater levels of ROS were produced by APE1-deficient human gastric and colonic cell lines and primary gastric epithelial cells compared to control cells after infection with either gastric or enteric pathogens. H. pylori activated Rac1 and Nox1 in all cell types, but activation was higher in APE1 suppressed cells. APE1 overexpression decreased H. pylori-induced ROS generation, Rac1 activation, and Nox1 expression. We determined that the effects of APE1 were mediated through its N-terminal lysine residues interacting with Rac1, leading to inhibition of Nox1 expression and ROS generation. APE1 is a negative regulator of oxidative stress in the gastrointestinal epithelium during bacterial infection by modulating Rac1 and Nox1. Our results implicate APE1 in novel molecular interactions that regulate early stress responses elicited by microbial infections.

[Diagnostic validity of fecal occult blood test in infants with food protein-induced allergic proctocolitis]. / Estudio de validez diagnóstica de la prueba de hemorragia oculta fecal en lactantes con proctocolitis alérgica inducida por proteína alimentaria.

INTRODUCTION: Food protein-induced allergic proctocolitis (FPIAP) is the most frequent presenta tion of non-IgE mediated food allergy (FA). The diagnosis is made by oral food challenge, however, non-invasive diagnostic tests are not available. In Chile, the fecal occult blood test (FOBT) is fre quently used to confirm FPIAP, however, there are no studies that support this practice. OBJECTIVE: To establish the diagnostic validity of FOBT in the evaluation of infants with FPIAP. PATIENTS AND METHOD: Case-control study with prospective recruitment of infants with rectal bleeding and suspicion of FPIAP, and controls were healthy infants, in whom the FOBT was conducted. All cases underwent an elimination diet, after which the diagnosis of FPIAP was confirmed by oral food cha llenge. RESULTS: 25 cases and 29 controls were included without significant differences in age, gen der, type of delivery, feeding, and maternal age. The cases had higher rates of allergic comorbidities, medication use, and family history of allergy. The FOBT was positive in 84% of cases and in 34% of controls (p < 0.001). The sensitivity of the FOBT for the diagnosis of FPIAP was 84%, specificity was 66%, positive predictive value 68%, and the negative predictive value 83%. The area under the ROC curve was 0.75 (CI 95% 0.61-0.88). CONCLUSIONS: Although the FOBT has an adequate sensitivity to diagnose FPIAP in infants with rectal bleeding, this test had abnormal results in more than a third of healthy infants. Therefore, the routine use of FOBT is not recommended for the diagnosis of FPIAP.

Helicobacter pylori-Clarithromycin Resistance in Symptomatic Pediatric Patients in a High Prevalence Country.

INTRODUCTION: Failure to eradicate Helicobacter pylori despite antibiotic treatment is generally attributed to increasing clarithromycin resistance conferred by point mutations in the 23S-rRNA gene or metronidazole resistance attributed to rdxA gene (HP0954) deletion in patients. Scarce data for pediatric population are available from developing countries. OBJECTIVES: The aim of the present study was to determine the presence of A2142G/C and A2143G mutations in the 23S-rRNA gene and/or rdxA gene (HP0954) deletion in a group of symptomatic H pylori-infected children recruited from an area with high infection rate and risk of gastric cancer. PATIENTS AND METHODS: We recruited 118 patients referred for upper endoscopy for gastrointestinal symptoms. The presence of H pylori was determined by urease test and histological staining. The rdxA gene (HP0954) deletion, and 2142G/C and A2143G mutations were determined by polymerase chain reaction-restriction fragment length polymorphism. A subgroup of infected patients received a 14-day regimen of omeprazole, amoxicillin, and clarithromycin. The effectiveness of this regime was determined by stool antigen determination 8 weeks after treatment. RESULTS: About 21% of the analyzed infected patients showed mutation in the 23S-rRNA gene, with the A2143G transition as the more frequent mutation, and 2% of the patients showed rdxA gene (HP0954) deletion. After treatment, 25% of the patients continued to harbor the bacteria; of these, 67% carried the A2143G mutation. CONCLUSIONS: H pylori-infected pediatric patients from Chile show high prevalence of the mutation responsible for clarithromycin resistance. The failure to eradicate H pylori can be attributed to the presence of the A2143G mutation.

Iron Deficiency and IL1ß Polymorphisms in Helicobacter pylori-infected Children.

BACKGROUND: Helicobacter pylori infection has been associated with an imbalance of iron homeostasis. IL-1ß has been related with iron absorption disturbances through a variety of mechanisms. The aim of this study was to evaluate the presence of polymorphic variants for IL-1ß cluster and gastric IL1ß mRNA expression in H. pylori-infected children and their relationship with hypochlorhydria and iron deficiency (ID). PATIENTS AND METHODS: Prospective study of 123 symptomatic children. At endoscopy, antral biopsies were taken for urease test, pathology and culture and blood for analysis of ferritin, transferrin, serum iron, and total iron-binding capacity. Polymorphisms in the IL-1ß cluster (positions -511, -31, +3954, ILRN) were determined by PCR-RFLP. Gastric mucosal expression of IL-1ß mRNA was determined by RT-PCR. RESULTS: After exclusions, of 105 patients, 33 (31.4%) were H. pylori positive. Nine (8.6%) children were classified as iron deficient (ID). Helicobacter pylori positivity was associated with ID (OR: 5.1; 95% CI: 1.2-21.9) (p = .04). No significant differences were found in allele frequency for IL1ß gene cluster polymorphisms between infected and uninfected children. Helicobacter pylori-infected children with ID had significantly increased gastric IL1ß mRNA in comparison with infected children without ID. In addition, a significant positive correlation was observed between mucosal IL-1ß mRNA and fasting gastric juice pH. Gastric pH values were significantly increased in H. pylori-infected patients with ID compared to uninfected children. CONCLUSIONS: The established association between H. pylori infection and ID in children may be mediated by increased gastric mucosal IL-1ß.

[Maternal infection due to Helicobacter pylori does not increase the risk of the infection in the first trimester of the life of their infants]. / La infección materna por Helicobacter pylori no aumenta el riesgo de contraer la bacteria en el primer trimestre de vida de sus lactantes.

INTRODUCTION: H. pylori infection is acquired early in childhood. However, there is little information available regarding the role of breastfeeding and neonatal acquisition of the infection. OBJECTIVE: To evaluate factors affecting the acquisition of H. pylori in newborns and infants from infected mothers. PATIENTS AND METHOD: Consecutive mothers and their newborns were recruited into the study from the maternity unit, immediately after delivery. After signing informed consent, one stool sample from the mother was obtained before hospital discharge. Three stool samples of the newborns were then collected at home at 15, 60, and 90 days of life, for the detection of H. pylori antigen (Monoclonal HpSAg, sensitivity 94% and specificity 97%). The socio-epidemiological and biomedical variables were also analysed using a questionnaire. RESULTS: A total of 32 mother-child pairs (64 subjects) were enrolled. The mean maternal age was 30.1±5.1 years, with 53% vaginal delivery, and 85% exclusively breastfed. There were 13 (40%) infected mothers. No H. pylori infection was detected in newborns and infants up to 3 months of follow-up. No significant differences were found in socioeconomic level between infected versus non-infected mothers (both groups mostly in the very high socioeconomic category: 28% and 32%, respectively, P=.15) and in the number of family members between infected versus non-infected mothers (3.8±0.8 vs 4.2±1.8 persons, P=.18). CONCLUSION: Despite having a significant percentage of H. pylori-infected mothers, no newborn was infected at the third month of life. The protective role of breastfeeding cannot be ruled out.

Serological response to Helicobacter pylori infection among Latin American populations with contrasting risks of gastric cancer.

Gastric cancer is a rare outcome of chronic Helicobacter pylori infection. Serologic profiles may reveal bacterial, environmental and/or host factors associated with cancer risk. We therefore compared specific anti-H. pylori antibodies among populations with at least twofold differences in gastric cancer mortality from Mexico, Colombia and Chile. Our study included 1,776 adults (mean age 42 years) from three nationally representative surveys, equally divided between residents of high- and low-risk areas. Antibodies to 15 immunogenic H. pylori antigens were measured by fluorescent bead-based multiplex assays; results were summarized to identify overall H. pylori seropositivity. We used logistic regression to model associations between antibody seroreactivity and regional cancer risk (high vs. low), adjusting for country, age and sex. Both risk areas had similar H. pylori seroprevalence. Residents in high- and low-risk areas were seroreactive to a similar number of antigens (means 8.2 vs. 7.9, respectively; adjusted odds ratio, OR: 1.02, p = 0.05). Seroreactivities to Catalase and the known virulence proteins CagA and VacA were each significantly (p < 0.05) associated with residence in high-risk areas, but ORs were moderate (1.26, 1.42 and 1.41, respectively) and their discriminatory power was low (area under the curve < 0.6). The association of Catalase was independent from effects of either CagA or VacA. Sensitivity analyses for antibody associations restricted to H. pylori-seropositive individuals generally replicated significant associations. Our findings suggest that humoral responses to H. pylori are insufficient to distinguish high and low gastric cancer risk in Latin America. Factors determining population variation of gastric cancer burden remain to be identified.

Brain angiogenesis inhibitor 1 is expressed by gastric phagocytes during infection with Helicobacter pylori and mediates the recognition and engulfment of human apoptotic gastric epithelial cells.

After Helicobacter pylori infection in humans, gastric epithelial cells (GECs) undergo apoptosis due to stimulation by the bacteria or inflammatory cytokines. In this study, we assessed the expression and function of brain angiogenesis inhibitor 1 (BAI1) in the engulfment of apoptotic GECs using human tissue and cells. After induction of apoptosis by H. pylori or camptothecin, there was a 5-fold increase in the binding of apoptotic GECs to THP-1 cells or peripheral blood monocyte-derived macrophages as assayed by confocal microscopy or conventional and imaging flow cytometry. Binding was impaired 95% by pretreating apoptotic cells with annexin V, underscoring the requirement for phosphatidylserine recognition. The phosphatidylserine receptor BAI1 was expressed in human gastric biopsy specimens and gastric phagocytes. To confirm the role of BAI1 in apoptotic cell clearance, the functional domain of BAI1 was used as a competitive inhibitor or BAI1 expression was inhibited by small interfering RNA. Both approaches decreased binding and engulfment >40%. Exposing THP-1 cells to apoptotic cells inhibited IL-6 production from 1340 to <364 pg/ml; however, this decrease was independent of phagocytosis. We conclude that recognition of apoptotic cells by BAI1 contributes to their clearance in the human gastric mucosa and this is associated with anti-inflammatory effects.

Helicobacter pylori infection inhibits phagocyte clearance of apoptotic gastric epithelial cells.

Increased apoptotic death of gastric epithelial cells is a hallmark of Helicobacter pylori infection, and altered epithelial cell turnover is an important contributor to gastric carcinogenesis. To address the fate of apoptotic gastric epithelial cells and their role in H. pylori mucosal disease, we investigated phagocyte clearance of apoptotic gastric epithelial cells in H. pylori infection. Human gastric mononuclear phagocytes were analyzed for their ability to take up apoptotic epithelial cells (AECs) in vivo using immunofluorescence analysis. We then used primary human gastric epithelial cells induced to undergo apoptosis by exposure to live H. pylori to study apoptotic cell uptake by autologous monocyte-derived macrophages. We show that HLA-DR(+) mononuclear phagocytes in human gastric mucosa contain cytokeratin-positive and TUNEL-positive AEC material, indicating that gastric phagocytes are involved in AEC clearance. We further show that H. pylori both increased apoptosis in primary gastric epithelial cells and decreased phagocytosis of the AECs by autologous monocyte-derived macrophages. Reduced macrophage clearance of apoptotic cells was mediated in part by H. pylori-induced macrophage TNF-α, which was expressed at higher levels in H. pylori-infected, compared with uninfected, gastric mucosa. Importantly, we show that H. pylori-infected gastric mucosa contained significantly higher numbers of AECs and higher levels of nonphagocytosed TUNEL-positive apoptotic material, consistent with a defect in apoptotic cell clearance. Thus, as shown in other autoimmune and chronic inflammatory diseases, insufficient phagocyte clearance may contribute to the chronic and self-perpetuating inflammation in human H. pylori infection.

Peptic ulcer disease in Helicobacter pylori-infected children: clinical findings and mucosal immune response.

OBJECTIVES: Peptic ulcer disease (PUD) is highly prevalent among adults but less common in children. Helicobacter pylori infection, the main cause of PUD, is, however, acquired extremely early in life. The aim of the study was to analyze clinical characteristics of children with PUD in a country with a high prevalence of the disease and to evaluate which host factors could determine this clinical outcome. METHODS: Children referred for upper gastrointestinal (GI) endoscopy with suspicion of peptic diseases were included prospectively during an 8-year period. Antral biopsies were performed to determine H pylori presence and mucosal cytokines profile. RESULTS: A total of 307 children between 3 and 18 years old were enrolled. Of the total, 237 children (46% boys) with complete data were included. H pylori infection was confirmed in 133 (56.1%) participants. Duodenal ulcer (DU) was diagnosed in 32 patients (13.5%); among them 29 were infected with H pylori (90.6%). Infected children had a nodular appearance of the gastric mucosa more often than noninfected children. Noninfected children had fewer lymphoid follicles and less inflammatory infiltrate than infected children. Only mucosal polymorphonuclear cell infiltration was more intense in DU-infected children as compared with non-DU-infected children. DU-infected children had higher levels of mucosal interferon-γ than noninfected and non-DU-infected patients. Non-DU-infected children had also higher levels of mucosal interleukin-10 than noninfected patients (P < 0.05). CONCLUSIONS: PUD in children, especially DU, is strongly associated with H pylori infection in developing countries. There is no distinctive clinical presentation of children with PUD. T-helper cytokine balance may influence clinical outcomes in children.

[Crigler-Najjar syndrome. Report of one case with a long term follow up]. / Evolución prolongada en síndrome de Crigler-Najjar tipo I.

Crigler-Najjar Syndrome is an uncommon genetic disorder characterized by the elevation of unconjugated plasmatic bilirubin secondary to deficiency of the enzyme uridine diphosphate glucuronyltransferase (UDP-GT). We report a 19-years-old woman with the syndrome diagnosed during the neonatal period, when she developed a severe jaundice in the first 10 days of life, reaching unconjugated bilirubin levels of 29 mg/dl, with normal liver function tests. After transient response to phototherapy, the patient was referred to a tertiary medical center in which an extensive work up ruled out other etiologies and the diagnosis of type I Crigler-Najjar syndrome was established. Currently, the patient has a mild mental retardation. She is receiving homemade phototherapy 18 h per day with acceptable control of bilirubin levels. Many mutations have been associated with UDP-GT dysfunction resulting in a broad spectrum of the disease. When bilirubin rises above physiological limits, it permeates the hematoencephalic barrier, inducing bilirubin impregnation of basal ganglia with secondary neuronal damage and necrosis. The worst outcome, kernicterus, is characterized by mental retardation, central deafness, ophthalmoplegia, ataxia, athetosis, spasticity, seizures and death. First line therapy includes phototherapy, but definitive therapy is liver transplantation before the occurrence of neurological damage.