Objective: Treatment compliance in children with Crohn’s disease is associated with higher levels of symptom remission. We hypothesized that the management, comorbidities, and complications for children with Crohn’s disease would differ based on a diagnosis of noncompliance. Methods: Using the Kids’ Inpatient Database for 2006-2012, we identified young patients (<21 >years) with a diagnosis of Crohn’s disease. Diagnoses and procedures were analyzed according to a recorded diagnosis of noncompliance. Multivariable logistic regression analysis was performed to examine the association between noncompliance and the outcomes of interest. Results: There were 28,337 pediatric Crohn’s disease hospitalizations identified with 1,028 (3.6%) hospitalizations having a diagnosis of both Crohn’s disease and noncompliance. The mean age of the study population was 15.9 years and 48.9% were girls. Black patients ( multivariable adjusted odds ratio, aOR,2.27; 95% CI:1.84-2.79) and those in the lowest income quartile (aOR 1.57; 95% CI:1.20-2.05) had an increased likelihood of a noncompliance diagnosis than respective comparison groups. Noncompliant patients had an increased likelihood of concurrent depression, nutritional deficiency, and anemia. Patients with a diagnosis of noncompliance had lower rates of intestinal obstruction (4.0% vs 6.3%), intraabdominal abscesses (2.0% vs 4.2%,), and underwent fewer major surgical procedures (aOR 0.40; 95% CI:0.31-0.53) and large bowel resections (aOR 0.44; 95% CI:0.31-0.64) than patients without this diagnosis. Conclusions: We found significant differences in socioeconomic status and race among hospitalized children with Crohn’s disease with, as compared to those without, a diagnosis of noncompliance. Children with noncompliance have different comorbidities, disease-related complications, and are managed differently. Possible explanations for observed treatment differences include a reluctance to offer surgery to those with a diagnosis of noncompliance, a refusal of intervention by noncompliant patients, or implicit bias. Further investigation is warranted to better define noncompliance in this population and to determine the implications of this diagnosis.

Nonalcoholic steatohepatitis (NASH) is a severe liver disorder characterized by triglyceride accumulation, severe inflammation, and fibrosis. With the recent increase in prevalence, NASH is now the leading cause of liver transplantation, with no approved therapeutics available. Despite years of research, the exact molecular mechanism of NASH progression is not well understood, but fat accumulation is believed to be the primary driver of the disease. Therefore, diacylglycerol O-acyltransferase 2 (DGAT2), a key enzyme in triglyceride synthesis, has been explored as a NASH target. RNAi-based therapeutics is revolutionizing the treatment of liver diseases, with recent chemical advances supporting long term gene silencing with single subcutaneous administration. Here we identified a hyper-functional, fully chemically stabilized GalNAc conjugated siRNA targeting DGAT2 (Dgat2-1473) that upon injection elicits up to three months of DGAT2 silencing (>80-90%, p<0.0001) in wild-type and NSG-PiZ “humanized” mice. Using an obesity-driven mouse model of NASH (ob/ob-GAN), Dgat2-1473 administration prevents and reverses triglyceride accumulation (> 50%, p:0.0008), resulting in significant improvement of the fatty liver phenotype. However, surprisingly, the reduction in liver fat didn’t translate into a similar impact on inflammation and fibrosis. Thus, while Dgat2-1473 is a practical, long-lasting silencing agent for potential therapeutic attenuation of liver steatosis, combinatorial targeting of a second pathway may be necessary for therapeutic efficacy against NASH.

Adipose tissue inflammation has an impact on liver health and it has been demonstrated that chronic alcohol consumption leads to the expression of pro-inflammatory markers in the adipose tissue. A thorough characterization of alcohol-induced adipose inflammation is lacking, and is important to understand in order to identify immune-related mechanisms that drive this phenomenon. Current therapeutic regimens for alcoholic liver disease are ineffective. It is critical to understand how other organs influence liver injury in this disease when developing novel and effective therapies in the future. Alcoholic liver disease exhibits a sexual dimorphism; women are more susceptible to liver injury than men and the same paradigm exists in rodent models. Here, I demonstrate that female mice have greater alcohol-induced adipose tissue inflammation than male mice, evidenced by greater expression of pro-inflammatory cytokines and cell markers. Further, female mice also exhibit higher expression of toll-like receptor genes in the adipose tissue, suggesting a potential role for the innate immune system in alcohol-induced adipose inflammation. Toll-like receptor 4 (TLR4) has been demonstrated to drive inflammation in both the liver and adipose tissue. I used both germline and conditional knockouts of Tlr4 to characterize alcohol-induced changes in the immune cell composition of adipose tissue. Alcohol increased the number of pro-inflammatory adipose tissue macrophages. This macrophage phenotype switching is partially dependent on TLR4; germline, but not myeloid-specific, Tlr4-deletion prevents macrophage phenotype switching. Overall, my work demonstrates that alcohol-induced adipose tissue inflammation is related to liver injury and that TLR4 contributes to adipose macrophage phenotype switching.

Chronic intake of alcohol can result in a range of pathology in the liver. Whilst the earliest changes observed with chronic ethanol, including the accumulation of lipid, or steatosis, are readily reversible upon cessation of alcohol consumption, longer exposure to ethanol may achieve more complex disease states including steatohepatitis, fibrosis, and cirrhosis that can cause irreversible damage and progress to fulminant hepatic failure. A key concept in the pathogenesis of alcoholic liver disease is that chronic ethanol primes the liver to increased injury through an interplay between hepatocytes and non-parenchymal cells, chiefly immune cells, of the liver. These relationships between hepatocytes and non-parenchymal cell types in alcoholic liver disease are reviewed in Chapter 1A. The Hypoxia Inducible Factors are a set of transcription factors that classically have been described as affecting a homeostatic response to conditions of low oxygen tension. Alcoholic liver disease is marked by increased hepatic metabolic demands, and some evidence exists for increased hepatic tissue hypoxia and upregulation of hypoxia-inducible factor mRNA with chronic alcohol. However, the biological significance of these findings is unknown. In Chapter 1B, we review the literature on recent investigations on the role of hypoxia inducible factors in a broad array of liver diseases, seeking to find common themes of biological function. In subsequent chapters, we investigate the hypothesis that a member of the hypoxia inducible- factor family, HIF1α, has a role in the pathogenesis of alcoholic liver disease. In Chapter 2, we establish a mouse model of alcoholic liver disease and report data confirming HIF1α activation with chronic ethanol. We demonstrate that HIF1α protein, mRNA, and DNA binding activity is upregulated in ethanol-fed mice versus pair-fed mice, and that some upregulation of HIF2α protein is observable as well. In Chapter 3, we utilize a mouse model of hepatocyte-specific HIF1α activation and demonstrate that such mice have exacerbated liver injury, including greater triglyceride accumulation than control mice. Using cre-lox technology, we introduce a degradation resistant mutant of HIF1α in hepatocytes, and after four weeks of ethanol feeding, we demonstrate that mice with the HIF1α transgene have increased liver-weight to body weight ratio and higher hepatic triglyceride levels. Additionally, several HIF1α target genes are upregulated. In Chapter 4, we examine the relationship between HIF1α activation and hepatic lipid accumulation using a recently published in vitro system, in which lipid accumulation was observed after treating Huh7 cells with the chemokine Monocyte Chemoattractant Protein-1 (MCP-1). We report that MCP-1 treatment induces HIF1α nuclear protein accumulation, that HIF1α overexpression in Huh7 cells induces lipid accumulation, and finally, that HIF1α siRNA prevents MCP-1 induced lipid accumulation. In Chapter 5, we use mouse models to investigate the hypothesis that suppression of HIF1α in hepatocytes or cells of the myeloid lineage may have differing effects on the pathogenesis of alcoholic liver disease. We find that ethanol-fed mice expressing a hepatocyte-specific HIF1α deletion mutant exhibit less elevation in liver-weight body ratio and diminished hepatic triglycerides versus wild-type mice; furthermore, we find that challenging these mice with lipopolysaccharide (LPS) results in less liver enzyme elevation and inflammatory cytokine secretion than in wild-type mice. In Chapter 6, we offer a final summary of our findings and some directions for future work.

Blood glucose (BG) spikes increase chronic disease risk in those with and without diabetes. The fiber and phytonutrient content of fruit, such as raspberries has the potential to attenuate the BG spike of a high glycemic load (HGL) meal. We examined the effect of red raspberries (RRs) on the BG spike when added to a HGL meal. In this randomized cross-over trial, 16 university students were given breakfast cereal and almond milk (control), the control breakfast with 140g added RRs, and the control breakfast with sugar equivalent to the sugar content of the RRs (50% sugar increase from control). Meals given on 3 separate test days, being separated by a 1-week washout period. BG was measured at: fasting then 15 min, 30 min, 60 min, 90 min, and 120 min postprandial. Repeated measures ANOVA was performed to investigate differences in peak minus baseline BG (spike) and glucose incremental area under the curve (IAUC). The added RR meal had a lower BG spike compared to the added sugar meal (-12.8 mg/dL, CI: -22.8 to -2.79, p = 0.0083). Additionally, a lower BG was seen comparing the added RR meal to the control meal (-10.9 mg/dL, CI: -20.9 to -0.83, p = 0.0318). There were no significant differences for IAUC and the timing of peak BG among the meals. RR effect is likely attributed to high fiber-to-sugar ratio, polyphenol content, and greater amount of beneficial micronutrients. Based on obtained results from this pilot study, the practice of adding RRs to HGL meals such as breakfast, seems to reduce risk for T2DM in health populations through BG spike attenuation.

Clostrioides difficile is a common cause of nosocomial (hospital-acquired) infections. Patients receiving antibiotic treatment experience dysbiosis of gut microbiota, and C. difficile, normally held in check by the various other organisms, takes this opportunity to propagate. Symptoms of infection generally include diarrhea, colitis, dehydration, and fever. Understanding that C. difficile generally only causes illness when it is the dominant bacterium (i.e. when growth is relatively unchecked by other microbes), it is appropriate to investigate potential competitive organisms that may be introduced after antibiotic courses or during active C. difficile infection to effectively displace it. Fecal samples from the University of Central Florida Lift fecal collection station were aseptically plated onto modified cycloserine cefoxitin fructose agar (CCFA). Visually remarkable colonies (certain colonies that looked unique in comparison to others) were restreaked on new plates of the same media to verify growth, then transferred to brain heart infusion-supplemented (BHIS) plates for propagation. Colonies were inoculated in glycerol stocks for storage, then grown in BHIS liquid media to prepare for identification. Genomic extraction was performed on each sample, and spectrophotometric quantification and gel electrophoresis were executed to confirm successful extraction. Genomic samples will be sent to an external laboratory for identification via polymerase chain reaction and Sanger sequencing. We hypothesize that at least one bacterial strain from the fecal collection station will potentially inhibit C. difficile infection. Should such an organism be identified, it follows that the efficacy of its application in conventional hospital settings may be examined. Current regulation of fecal microbiota transplants, an effective therapeutic practice, is cumbersome, and changing the classification of fecal transplants may improve timeliness and effectiveness of treatment.

The regulation of glucagon-like peptide-1 and peptide YY hormone levels are regulated based on different influential factors, but primarily levels are dependent upon ingested food content. As meals today become more fat-enriched, there is greater requirement for evaluation of these hormones that regulate insulin and satiety levels within the body. We have shown that the gene expression transcript production of glucagon-like peptide-1 and peptide YY are modulated by different concentrations, and times of short-chain fatty acids and long-chain fatty acids. Although the peptide hormone levels have the influential physiological role on effector tissue, the regulation of these hormones begins at the transcript levels. Recent research indicates that glucagon-like peptide-1 and peptide YY hormones are altered in response to different free-fatty acids. The present investigation generally demonstrated an overall decrease in both hormones after chronic exposure to fatty acids. Intestinal secretin tumor cell line (STC-1 cells) was used as a representative for intestinal L-cells. Quantitative real-time PCR analysis was used to determine the changes in RNA transcripts. Overall, there was a decrease in the 3-hour timeline, which continued to decrease in the 16-hour and 24-hour timelines for glucagon-like peptide-1. Peptide YY transcript expression in 3-hours increased significantly after exposure to propionate, a significant decrease after exposure to acetate, and no significant increase or decrease after exposure to butyrate. However, there was a significant decrease in peptide YY once reaching 24-hour exposure. It was determined there is a threshold for different concentrations of free-fatty acids to influence glucagon-like peptide-1 and peptide YY production, which was present in the different concentrations of butyrate. Lastly, exposure to both concentrations of linolenic acid caused a significant decrease in glucagon-like peptide-1 and peptide YY.

C1q TNF Related Protein 3 (CTRP3), is a cytokine that is primarily secreted from adipose tissue, which classifies it as an adipokine. Our previous research has shown that CTRP3 prevents alcoholic fatty liver disease (ALD) in male mice. However, even when accounting for confounding factors such as absolute and relative alcohol intake, females are more sensitive to the effects of consumption compared to male mice. Therefore, the goal of this project was to determine whether CTRP3 prevented ALD in female mice. Methods: Female wild type (WT) and female CTRP3 transgenic over expressing (Tg) mice were fed an ethanol containing liquid diet (5% v/v) for 6 weeks. Daily weight and food intake measurements were taken and external heat-pads were placed under a portion of the cage to facilitate thermoregulation. Hepatic steatosis was determined by total triglyceride quantification and lipid droplet quantitation in liver sections. Data were analyzed by repeated measures ANOVA, t-test, or Log-rank (Mantel-Cox) test as appropriate. Results: There was no difference between WT and Tg mice in food intake or body weight. There was no difference in survival between WT and Tg mice, however, Tg mice trended towards a reduced rate of survival compared with WT mice (78% in WT versus 44% in Tg, p=0.13). Stereological analysis indicated no difference in the percent of lipid liver volume between the two groups (WT 7.2±3.6 vs Tg 5.1±4.1%). This finding was consistent with no difference in total hepatic triglyceride accumulation observed between WT and Tg mice (12.7±4.4 vs. 13.1±6.8 mg triglycerides/gram liver protein). Conclusion: Combined these data indicate that unlike previous studies with male mice, CTRP3 is not protective against alcohol-induced hepatic steatosis in female mice. Combined, these data indicate that the adipokines such as CTRP3 contribute to physiology in a sex-specific manner.

Background: Recent national trends and distribution of ileal pouch-anal anastomosis (IPAA) procedures for patients with ulcerative colitis (UC) are unknown. We examined the frequency of use of elective IPAA procedures among patients with UC and the distribution of IPAA procedures across more than 140 U.S. academic medical centers and their affiliates. Methods: Data were obtained from the University HealthSystem Consortium for patients with a primary diagnosis of UC admitted electively between 2012 and 2015. Results: The mean age of the study population (n=6,875) was 43 years and 57% were men. Among these, one-third (n=2,307) underwent an IPAA, while two-thirds (n=4,568) underwent colectomy, proctectomy, proctocolectomy or other procedures. The proportion of IPAA cases among all elective admissions was relatively stable at 33-35% during the years under study. A total of 131 hospitals, out of 279 hospitals participating in the UHC, performed IPAA. The median number of IPAA cases performed annually was 1.9 [IQR 0.8 – 4.3]. Nearly one half (48%) of these cases were performed by the top ten hospitals. Overall, only a total of 30 centers performed ³ five elective IPAA cases annually. Conclusions: Although the frequency of elective IPAA surgery in recent years has been stable, nearly one half of all IPAA cases was performed at ten hospitals. The concentration of IPAA cases at high-volume centers, and the steady number of cases performed annually, have potential implications for fellowship training, patient clinical outcomes and access to care.

Background: Treatment decisions for elderly patients with gallbladder pathology are complex. Little is known about what factors go into treatment decisions in this population. We used Medicare data to examine gender-based differences in the use of cholecystectomy vs. cholecystostomy tube placement in elderly patients with acute cholecystitis. Methods: We queried a 5% random sample of Medicare data (2009-2011) for patients >65 admitted for acute cholecystitis (by ICD-9 code) who subsequently underwent a cholecystectomy and/or cholecystostomy tube placement. Demographic information (age, race), clinical characteristics (Elixhauser index, presence of biliary pathology), and hospital outcomes (case fatality rate, length of stay, need for ICU care) were compared by gender. A multivariable model was used to examine predictors of cholecystectomy vs. cholecystostomy tube placement. Results: Of 4063 patients admitted with cholecystitis undergoing the procedures of interest just over half (58%) were women. The majority of patients (93%) underwent cholecystectomy. Compared to women, men were younger (average age 76 vs. 78, p value < 0.01) and had few comorbidities (average Elixhauser 1.2 vs. 1.4 p value < 0.01). Case fatality rate was similar between men (2.5%) and women (2.4% p value 0.48). A higher percentage of men spent time in the ICU (36%) compared to women (31% p value < 0.01). On multivariable analysis men were 30% less likely to undergo cholecystectomy (OR 0.69, 95% CI 0.53-0.91). Conclusion: Elderly men are less likely than elderly women to undergo cholecystectomy for acute cholecystitis despite being younger with less co morbidity and are more likely to spend time in the ICU. More research is needed to determine whether a difference in treatment is contributing to the higher rate of ICU utilization in elderly men with acute cholecystitis.