Deborah Schady

Novel FOXF1 Mutations in Sporadic and Familial Cases of Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins Imply a Role for its DNA Binding Domain

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare and lethal developmental disorder of the lung defined by a constellation of characteristic histopathological features. Nonpulmonary anomalies involving organs of gastrointestinal, cardiovascular, and genitourinary systems have been identified in approximately 80% of patients with ACD/MPV. We have collected DNA and pathological samples from more than 90 infants with ACD/MPV and their family members. Since the publication of our initial report of four point mutations and 10 deletions, we have identified an additional 38 novel nonsynonymous mutations of FOXF1 (nine nonsense, seven frameshift, one inframe deletion, 20 missense, and one no stop). This report represents an up to date list of all known FOXF1 mutations to the best of our knowledge. Majority of the cases are sporadic. We report four familial cases of which three show maternal inheritance, consistent with paternal imprinting of the gene. Twenty five mutations (60%) are located within the putative DNA‐binding domain, indicating its plausible role in FOXF1 function. Five mutations map to the second exon. We identified two additional genic and eight genomic deletions upstream to FOXF1. These results corroborate and extend our previous observations and further establish involvement of FOXF1 in ACD/MPV and lung organogenesis.

New generation lipid emulsions prevent PNALD in chronic parenterally fed preterm pigs

Total parenteral nutrition (TPN) is associated with the development of parenteral nutrition-associated liver disease (PNALD) in infants. Fish oil-based lipid emulsions can reverse PNALD, yet it is unknown if they can prevent PNALD. We studied preterm pigs administered TPN for 14 days with either 100% soybean oil (IL), 100% fish oil (OV), or a mixture of soybean oil, medium chain triglycerides (MCTs), olive oil, and fish oil (SL); a group was fed formula enterally (ENT). In TPN-fed pigs, serum direct bilirubin, gamma glutamyl transferase (GGT), and plasma bile acids increased after the 14 day treatment but were highest in IL pigs. All TPN pigs had suppressed hepatic expression of farnesoid X receptor (FXR), cholesterol 7-hydroxylase (CYP7A1), and plasma 7α-hydroxy-4-cholesten-3-one (C4) concentrations, yet hepatic CYP7A1 protein abundance was increased only in the IL versus ENT group. Organic solute transporter alpha (OSTα) gene expression was the highest in the IL group and paralleled plasma bile acid levels. In cultured hepatocytes, bile acid-induced bile salt export pump (BSEP) expression was inhibited by phytosterol treatment. We show that TPN-fed pigs given soybean oil developed cholestasis and steatosis that was prevented with both OV and SL emulsions. Due to the presence of phytosterols in the SL emulsion, the differences in cholestasis and liver injury among lipid emulsion groups in vivo were weakly correlated with plasma and hepatic phytosterol content.

Targeted p16Ink4a epimutation causes tumorigenesis and reduces survival in mice

Cancer has long been viewed as a genetic disease; however, epigenetic silencing as the result of aberrant promoter DNA methylation is frequently associated with cancer development, suggesting an epigenetic component to the disease. Nonetheless, it has remained unclear whether an epimutation (an aberrant change in epigenetic regulation) can induce tumorigenesis. Here, we exploited a functionally validated cis-acting regulatory element and devised a strategy to induce developmentally regulated genomic targeting of DNA methylation. We used this system to target DNA methylation within the p16(Ink4a) promoter in mice in vivo. Engineered p16(Ink4a) promoter hypermethylation led to transcriptional suppression in somatic tissues during aging and increased the incidence of spontaneous cancers in these mice. Further, mice carrying a germline p16(Ink4a) mutation in one allele and a somatic epimutation in the other had accelerated tumor onset and substantially shortened tumor-free survival. Taken together, these results provide direct functional evidence that p16(Ink4a) epimutation drives tumor formation and malignant progression and validate a targeted methylation approach to epigenetic engineering.

Postnatal epigenetic regulation of intestinal stem cells requires DNA methylation and is guided by the microbiome

BackgroundDNA methylation is an epigenetic mechanism central to development and maintenance of complex mammalian tissues, but our understanding of its role in intestinal development is limited.ResultsWe use whole genome bisulfite sequencing, and find that differentiation of mouse colonic intestinal stem cells to intestinal epithelium is not associated with major changes in DNA methylation. However, we detect extensive dynamic epigenetic changes in intestinal stem cells and their progeny during the suckling period, suggesting postnatal epigenetic development in this stem cell population. We find that postnatal DNA methylation increases at 3′ CpG islands (CGIs) correlate with transcriptional activation of glycosylation genes responsible for intestinal maturation. To directly test whether 3′ CGI methylation regulates transcription, we conditionally disrupted two major DNA methyltransferases, Dnmt1 or Dnmt3a, in fetal and adult intestine. Deficiency of Dnmt1 causes severe intestinal abnormalities in neonates and disrupts crypt homeostasis in adults, whereas Dnmt3a loss was compatible with intestinal development. These studies reveal that 3′ CGI methylation is functionally involved in the regulation of transcriptional activation in vivo, and that Dnmt1 is a critical regulator of postnatal epigenetic changes in intestinal stem cells. Finally, we show that postnatal 3′ CGI methylation and associated gene activation in intestinal epithelial cells are significantly altered by germ-free conditions.ConclusionsOur results demonstrate that the suckling period is critical for epigenetic development of intestinal stem cells, with potential important implications for lifelong gut health, and that the gut microbiome guides and/or facilitates these postnatal epigenetic processes.

Serial Fecal Microbiota Transplantation Alters Mucosal Gene Expression in Pediatric Ulcerative Colitis

Serial Fecal Microbiota Transplantation Alters Mucosal Gene Expression in Pediatric Ulcerative Colitis

Characterizing pilomatricomas in children: a single institution experience

BACKGROUND/PURPOSE Pilomatricomas, or calcifying epitheliomas of Malherbe, are among the most common superficial cutaneous soft tissue lesions in children. Familiarity with the presenting signs and symptoms allows for the diagnosis to be made on physical examination alone in most patients, avoiding expensive and unnecessary diagnostic imaging. METHODS A retrospective IRB-approved review of surgical pathology archives and medical records of all patients undergoing excision of pilomatricomas between 1982 and 2010 was performed to determine the characteristics of the pilomatricoma tumors. Data regarding gender, age, location, size of tumor, and histopathology were collected. RESULTS There were 916 pilomatricomas resected in 802 patients. Fifty-five percent of the patients were girls (441 patients). The median age at the time of resection was 6 years (range 5 months to 18 years). Multiple lesions were found in 43 patients (5%). The most common location was head and neck (n = 529, 58%), followed by upper limbs (n = 214, 23%), trunk (n = 130, 14%), and lower limbs (n = 43, 5%). Information on size was available for 674 lesions; mean lesion diameter was 14.0 ± 7.4 mm. Twenty-eight patients (3%) had either recurrent (n = 11) or metachronous (n = 17) lesions resected at our institution, with a median interval of 12 months after initial resection (range 5 weeks to 5 years). No cases of pilomatrix carcinoma were observed. CONCLUSION The majority of pilomatricomas occur in the head and neck, although they can present in any location. Approximately 5% of children have multiple lesions. Pilomatricomas occur slightly more commonly in girls, and 66% of lesions occur in children < 10 years of age. Complete surgical excision is necessary to prevent recurrence. Recurrences and pilomatrix carcinoma are very rare if complete excision is achieved.

Streptococcus gallolyticus subsp. gallolyticus promotes colorectal tumor development

Streptococcus gallolyticus subsp. gallolyticus (Sg) has long been known to have a strong association with colorectal cancer (CRC). This knowledge has important clinical implications, and yet little is known about the role of Sg in the development of CRC. Here we demonstrate that Sg promotes human colon cancer cell proliferation in a manner that depends on cell context, bacterial growth phase and direct contact between bacteria and colon cancer cells. In addition, we observed increased level of β-catenin, c-Myc and PCNA in colon cancer cells following incubation with Sg. Knockdown or inhibition of β-catenin abolished the effect of Sg. Furthermore, mice administered with Sg had significantly more tumors, higher tumor burden and dysplasia grade, and increased cell proliferation and β-catenin staining in colonic crypts compared to mice receiving control bacteria. Finally, we showed that Sg is present in the majority of CRC patients and is preferentially associated with tumor compared to normal tissues obtained from CRC patients. These results taken together establish for the first time a tumor-promoting role of Sg that involves specific bacterial and host factors and have important clinical implications.

Primitive Myxoid Mesenchymal Tumor of Infancy Involving Chest Wall in an Infant: A Case Report and Clinicopathologic Correlation

Primitive myxoid mesenchymal tumor of infancy (PMMTI) is a rare mesenchymal tumor of early childhood characterized by aggressive local infiltration of surrounding structures, rare metastases, and poor response to chemotherapy. Surgery alone appears to be the most effective treatment given the lack of predilection for metastasis and poor response to traditional chemotherapy. Below we report a patient with PMMTI successfully managed with surgery and observation and summarize the existing literature on histopathologic features and treatment of this lesion.

Tu1192 Clinical, Epigenetic, and Metagenomic Responses to Serial Fecal Microbiome Transplants in Pediatric Ulcerative Colitis

G A A b st ra ct s and 4 mood-related questions. Emma provided correct answers. Emma v1 tested general knowledge and nutrition. Emma v2 featured more questions, 2 levels of difficulty, and was categorized by General, Anatomy & Testing, Medications, and Nutrition. We obtained information on sex, age group (10-14yr vs. 15-18yr), IBD-type, medications, and history of surgery. Feedback from patients and clinicians was obtained in both versions. Results: Sites 1 and 2 tested Emma v1 between February-August, and May-August 2013, respectively. Emma v2 was tested from November 1-November 23, 2013, and May-August 2013, respectively. A total of 58 patients (59% male, 67% Crohns) played Emma v1, while 33 patients (64% male, 58% Crohns) played Emma v2. Seven patients played both versions; only 7 patients played EMMA v2 at site 2. In Emma v1, 82% of disease-related questions were answered correctly. The mean mood scores for sadness (1.3, SD=0.69, range 1-4), anxiety (1.4, SD=0.9, range 1-5), energy (2.6, SD=1.5, range 1-5), and IBD-related QOL (2.0, SD= 1.5, range 1-5) indicated generally good functioning. In Emma v2, 61.1% of total knowledge questions were answered correctly, while percentages in General, Anatomy/Testing, Medications, and Nutrition were 66.8%, 65.8%, 74.5%, and 79.6%, respectively. Neither age nor IBD-type was correlated with disease knowledge. Of patients with difficult questions, 58.4% were answered correctly. The mean mood scores for sadness (1.6, SD=1.03, range 1-4), anxiety (2.2, SD=1.2, range 1-5), energy (2.2, SD=1.3, range 1-5), and IBD-related QOL (1.9, SD=1.3, range 1-5) indicated generally good functioning. Four patients were referred for mental health support. Conclusions: Pediatric patients with IBD need more diseaserelated education. The Emma ipad game has the potential to evaluate and teach gaps in knowledge, assess emotional functioning, and increase patient engagement.

Critical Role for the Microbiota in CX3CR1+ Intestinal Mononuclear Phagocyte Regulation of Intestinal T Cell Responses

Summary The intestinal barrier is vulnerable to damage by microbiota‐induced inflammation that is normally restrained through mechanisms promoting homeostasis. Such disruptions contribute to autoimmune and inflammatory diseases including inflammatory bowel disease. We identified a regulatory loop whereby, in the presence of the normal microbiota, intestinal antigen‐presenting cells (APCs) expressing the chemokine receptor CX3CR1 reduced expansion of intestinal microbe‐specific T helper 1 (Th1) cells and promoted generation of regulatory T cells responsive to food antigens and the microbiota itself. We identified that disruption of the microbiota resulted in CX3CR1+ APC‐dependent inflammatory Th1 cell responses with increased pathology after pathogen infection. Colonization with microbes that can adhere to the epithelium was able to compensate for intestinal microbiota loss, indicating that although microbial interactions with the epithelium can be pathogenic, they can also activate homeostatic regulatory mechanisms. Our results identify a cellular mechanism by which the microbiota limits intestinal inflammation and promotes tissue homeostasis. Graphical Abstract Figure. No caption available. HighlightsMicrobiota limits Th1 and supports Treg cell responses against intestinal antigensMicrobiota‐dependent anti‐inflammatory functions depend on CX3CR1+ MNPsMicrobiota supports CX3CR1+ MNP IL‐10 production that directs Th1 and Treg cell balanceCX3CR1+ MNP IL‐10 production requires microbial epithelial attachment &NA; Host microbiota interactions regulate many aspects of immunity. Kim et al. demonstrate that microbial adhesion to intestinal epithelium helps regulate the balance between pro‐ and anti‐inflammatory T cell responses through induction of IL‐10 by a subset of intestinal antigen‐presenting cells.