Judy Fuentebella

Increased Number of Regulatory T Cells in Children With Eosinophilic Esophagitis

Objectives: There are limited data on the role of regulatory T cells (Treg) in the disease pathology of eosinophilic esophagitis (EoE). We tested the differences in Treg in subjects with EoE compared with those with gastroesophageal reflux disease (GERD) and healthy controls (HC). Patients and Methods: Pediatric patients evaluated by endoscopy were recruited for our study. Participants were categorized into 3 groups: EoE, GERD, and HC. RNA purified from esophageal biopsies were used for real-time quantitative polymerase chain reaction assays and tested for forkhead box P3 (FoxP3) mRNA expression. Treg were identified as CD4+CD25hiCD127lo cells in peripheral blood and as CD3+/FoxP3+cells in esophageal tissue. Results: Forty-eight subjects were analyzed by real-time quantitative polymerase chain reaction: EoE (n = 33), GERD (n = 7), and HC (n = 8). FoxP3 expression was higher by up to 1.5-fold in the EoE group compared with the GERD and HC groups (P < 0.05). Protein levels of FoxP3 in blood and tissue were then investigated in 21 subjects: EoE (n = 10), GERD (n = 6), and HC (n = 5). The percentage of Treg and their subsets in peripheral blood were not significant between groups (P > 0.05). The amount of Treg in esophageal tissue was significantly greater in the EoE group (mean 10.7 CD3+/FoxP3+cells/high power field [HPF]) compared with the other groups (GERD, mean 1.7 CD3+/FoxP3+cells/HPF and HC, mean 1.6 CD3+/FoxP3+cells/HPF) (P < 0.05). Conclusions: We show that Treg are increased in esophageal tissue of EoE subjects compared with GERD and HC subjects. The present study illustrates another possible mechanism involved in EoE that implicates impairment of immune homeostasis.

Eotaxin and FGF enhance signaling through an Extracellular signal-related kinase (ERK)-dependent pathway in the pathogenesis of Eosinophilic Esophagitis

BackgroundEosinophilic esophagitis (EoE) is characterized by the inflammation of the esophagus and the infiltration of eosinophils into the esophagus, leading to symptoms such as dysphagia and stricture formation. Systemic immune indicators like eotaxin and fibroblast growth factor were evaluated for possible synergistic pathological effects. Moreover, blood cells, local tissue, and plasma from EoE and control subjects were studied to determine if the localized disease was associated with a systemic effect that correlated with presence of EoE disease.MethodReal-time polymerase chain reaction from peripheral blood mononuclear cells (PBMC), immunohistochemistry from local esophageal biopsies, fluid assays on plasma, and fluorescence-activated cell sorting on peripheral blood cells from subjects were used to study the systemic immune indicators in newly diagnosed EoE (n = 35), treated EoE (n = 9), Gastroesophageal reflux disease (GERD) (n = 8), ulcerative colitis (n = 5), Crohns disease (n = 5), and healthy controls (n = 8).ResultOf the transcripts tested for possible immune indicators, we found extracellular signal-regulated kinase (ERK), Bcl-2, bFGF (basic fibroblast growth factor), and eotaxin levels were highly upregulated in PBMC and associated with disease presence of EoE. Increased FGF detected by immunohistochemistry in esophageal tissues and in PBMC was correlated with low levels of pro-apoptotic factors (Fas, Caspase 8) in PBMC from EoE subjects. Plasma-derived bFGF was shown to be the most elevated and most specific in EoE subjects in comparison to healthy controls and disease control subjects.ConclusionWe describe for the first time a possible mechanism by which increased FGF is associated with inhibiting apoptosis in local esophageal tissues of EoE subjects as compared to controls. Eotaxin and FGF signaling pathways share activation through the ERK pathway; together, they could act to increase eosinophil activation and prolong the half-life of eosinophils in local tissues of the esophagus in EoE subjects.

Increased HLA-DR Expression on Tissue Eosinophils in Eosinophilic Esophagitis

Objective: The aim of the study was to investigate whether eosinophils have increased human leukocyte antigen (HLA)-DR expression in subjects with eosinophilic esophagitis (EoE) compared with controls. Patients and Methods: Patients who were undergoing an upper endoscopy with biopsies for suspected gastroesophageal reflux disease (GERD) or EoE at Lucile Packard Childrens Hospital were enrolled. In total, the blood and tissue samples of 10 healthy controls (HC), 11 subjects with GERD, and 10 with EoE were studied. Multiple tissue staining to identify eosinophils (via eosinophil cationic protein-clone EG2) and major histocompatibility complex class II cell surface receptors (via HLA-DR) was performed via immunohistochemistry. The peripheral blood was analyzed using flow cytometry to detect eosinophil HLA-DR expression among these subjects. Results: In the tissue, a greater proportion of eosinophils expressed HLA-DR among the subjects with EoE (mean 0.83 ± 0.14, n = 9) relative to those with GERD (mean 0.18 ± 0.19, n = 8, P < 0.01) and HC (mean 0.18 ± 0.13, n = 6, P < 0.01). In total, 6 participants (4 HC subjects and 2 subjects with GERD) did not have any eosinophils identified on tissue staining and were unable to be included in the present statistical analysis. In the blood, there was no statistically significant difference in eosinophil HLA-DR expression among HC subjects (mean 415 ± 217, n = 6), subjects with GERD (mean 507 ± 429, n = 2), and those with EoE (mean 334 ± 181, n = 6). Conclusions: These data demonstrate that the eosinophils from the esophagus of subjects with EoE have increased HLA-DR expression within this tissue.

Immunophenotyping of Peripheral Eosinophils Demonstrates Activation in Eosinophilic Esophagitis

Background and Aim: Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder characterized by upper gastrointestinal symptoms and the presence of high numbers of eosinophils in the esophagus. Although eosinophils in the esophagus have been found to be activated in subjects with EoE, detailed studies of intracellular signaling pathways involved in the mechanism of activation of eosinophils in EoE have heretofore been limited. The aim of the study was to assess whether any surface molecules or transcription factors are activated in peripheral eosinophils in subjects with EoE. Methods: Eosinophils and CD3+ lymphocytes were identified directly from 50 μL of whole blood of EoE and control subjects. Using Hi-FACS, levels of surface activation markers, including CD66b, and intracellular phosphoepitopes, including phosphorylated forms of signal transducer and activator of transcription (phospho-STAT) 1 and 6, were measured within each cell subset. Results: Levels of surface CD66b as well as levels of intracellular phospho-STAT1 and phospho-STAT6 in peripheral blood eosinophils were significantly higher for untreated subjects with EoE vs healthy controls (P < 0.05). Levels of phospho-STAT1 and phospho-STAT6 in peripheral blood eosinophils were lower in subjects with EoE on therapy versus untreated subjects with EoE (P < 0.05). Conclusions: Levels of phospho-STAT1 and phospho-STAT6, transcription factors involved in inflammatory processes, were both significantly higher in peripheral eosinophils from untreated (ie, newly diagnosed) subjects with EoE versus subjects with EoE on therapy, healthy controls. Blood-based measurements of CD66b and phospho-STAT levels in peripheral eosinophils may be beneficial for identifying EoE.

Nickel Toxicity Presenting as Persistent Nausea and Abdominal Pain

A previously healthy 13-year-old male presented to the pediatric gastroenterology clinic with persistent nausea and abdominal pain of 6-week duration. The pain was described as 3–5 out of 10 in severity, constant, and localized to the peri-umbilical area. The pain was aggravated by positional changes, application of pressure to the area, or by meals; it was somewhat relieved by assuming a fetal position or taking Pepto-Bismol. He was hungry but had early satiety; he would gag on occasion but had never vomited. His bowel movements were normal, 1–2 times a day, without blood or mucus. His parents were concerned because he had lost 6 lbs of weight in 6 weeks. He also had low-grade fevers, up to 102 F . He was initially treated with oseltamivir for 7 days on the presumption that he had influenza; however, his fevers persisted. He was subsequently diagnosed with sinusitis and was treated with an antibiotic whose name could not be recalled. After completion of a course of the antibiotic, he was treated with a second course of oseltamivir because of persistent fever and flu-like symptoms experienced by his brother. Thereafter, his fever curve began trending downward. However, amoxicillin was started because he was still having low-grade pyrexia. On review of systems, he had occasional temporal headaches. He had no cough, nasal congestion, respiratory difficulties, or chest pain. He had normal urinary habits, and no rash or joint pain or swelling. He would occasionally moan in his sleep and wake up complaining of hunger, but otherwise had no change in mental status or behavior. The remaining review of systems was unremarkable. He had no trauma or travel history. His past medical history was significant for pes planovalgus and joint hypermobility. He also had a history of failure to thrive when he was a toddler and was evaluated by a pediatric gastroenterologist at that time. An extensive work-up for his hypermobility and failure to thrive was negative. In 2005, he had presented with recurrent back pain and was found on ultrasound and magnetic resonance imaging (MRI) to have a gallstone and a retroperitoneal cyst; these findings led to a laparoscopic cholecystectomy and removal of the cyst. Pathologic examination of the cyst was reported as showing a benign epithelial lined cyst. Upon presentation, his only medication was amoxicillin, and he had no known drug or food allergies. Family history revealed that his brother had two episodes of spontaneous pneumothorax and was status postpleurodesis. The maternal side of the family had a history of gastric, liver, and brain cancers. On physical examination, he exhibited an asthenic habitus, had normal vital signs, and was in no acute distress. He was anicteric and had dark circles under his eyes. His oropharynx was clear, and he was wearing dental Section editor’s note: In an era of ever-increasing use of diagnostic laboratory and imaging tests, matched with frequent availability of tissue confirmation of the diagnosis, it is refreshing to read about a case that was solved on the basis of some forensic intelligence. This case serves as a subtle and humble reminder of the power of history and physical examination, matched by some ‘‘out of the box’’ thinking. Those of us looking after patients with chronic nausea and abdominal pain can very quickly appreciate the dramatically beneficial impact of the diagnosis that was made in this case by the pediatric gastroenterology team and how many long-term management issues were aborted. George Triadafilopoulos, MD. Editor, Stanford Multidisciplinary Seminars.

Abdominal pain, gastrointestinal bleeding, and weight loss in a 17-year-old male.

A 17-year-old male presented to his primary care physician with a 2-week history of epigastric pain. The pain was localized, burning, temporarily relieved by meals, and occasionally awakened him from sleep. He denied fever, chills, cough, vomiting, or changes in bowel habits. He also denied use of nonsteroidal anti-inflammatory drugs, illicit drugs, or alcohol. There was no history of travel or trauma. His physical examination was remarkable only for epigastric tenderness. Laboratory values including complete blood count (CBC), chemistry, liver panel, erythrocyte sedimentation rate (ESR) and serum Helicobacter pylori immunoglobulin G (IgG) were negative. He was empirically treated with omeprazole. However, his pain progressed in severity and he avoided oral intake. An abdominal ultrasound was normal. Upper endoscopy showed irregular, ulcerated folds extending from the gastroesophageal junction to the antrum (Fig. 1). Biopsies showed chronic active gastritis with poorly formed granulomas with central necrosis (Fig. 2). Hematoxylin and eosin (H&E) and Giemsa stains were negative for H. pylori. Acid fast bacilli (AFB), periodic acid-Schiff (PAS), and Gomori methenamine silver (GMS) stains were negative for mycobacteria and fungi. After having lost 10 pounds in 10 days, he was referred to our center for further evaluation. He developed hematemesis, which lowered his hematocrit level to 30.4%. ESR had elevated to 33 and C-reactive protein (CRP) was 7.6 mg/l. An urgent upper endoscopy revealed friable tissue, multiple nodules, ulcers throughout the stomach, and active bleeding. The esophagus and duodenum were normal. Biopsies showed poorly formed granulomas with central necrosis. Viral cultures, cytomegalovirus (CMV) polymerase chain reaction, AFB stain, and H. pylori were negative. Extensive workup, including tuberculin skin test, blood culture, rapid plasma reagin for syphilis, human immunodeficiency virus test, histoplasma antibody, screen Brucella antibody, antinuclear antibody, angiotensin-converting enzyme, chronic granulomatous disease test, and celiac panel were negative. Stool studies, including ova and parasites, culture, H. pylori antigen, and Clostridium difficile were negative. Chest X-ray was normal and abdominal computed tomography scan showed thickening and inflammation of the stomach and proximal duodenum. Upper gastrointestinal series with small bowel followthrough barium study showed mucosal disease in the distal esophagus, stomach, distal ileum, and proximal colon. Colonoscopy was normal except for lymphoid nodular hyperplasia. Biopsies were normal. He improved clinically with a protein pump inhibitor (PPI) and iron as his only medications. Three months later, he was seen in clinic for a follow-up visit. He remained asymptomatic, had regained his weight, and his hematocrit and ESR had normalized. He continued to take once daily PPI. A surveillance upper endoscopy showed normal mucosa throughout but biopsies showed J. Fuentebella (&) D. Bass Division of Gastroenterology, Hepatology, and Nutrition Department of Pediatrics, Lucile Packard Children’s Hospital, 750 Welch Road Suite 116, Palo Alto, CA 94304, USA e-mail: judyf@stanford.edu