Investigations Into Human Lipoxygenase Biosynthesis of Oxylipins, Their Roles in Inflammation, and Drug Discovery

The research in this dissertation explores the bioactivity and biosynthesis of lipoxygenase-derived products from polyunsaturated fatty acids as well as drug discovery. The diseases that cause most of the mortality today are due to chronic inflammation, which is caused by the lack of a proper resolution phase. The pro-resolution molecules are oxylipins catalyzed by oxygenases and derived from fish oil. Dietary, genetic, and environmental effects have a role in altering these lipid signaling pathways and causing inflammation. This thesis examines how these molecules are made, their effects on resolving inflammation, and possible relations to disease. The first chapter investigates human 12-lipoxygenase metabolites from omega-6 docosapentaenoic acid as a promising novel anti-platelet for the treatment of the life-threatening complications of cardiovascular diseases. The second chapter quantifies oxyipins and cytokines in human blood as possible biomarkers for heart failure. The next chapter characterizes the Pseudomonas Aeruginosa lipoxygenase, reveals possible roles in biofilm formation and manipulation of host immune response, and discovers first in class selective inhibitors. Finally, the last chapter describes the resolution of inflammation and the biosynthesis of a class of specialized proresolving mediators, the maresins

Investigations Into Human Lipoxygenase Biosynthesis of Oxylipins, Their Roles in Inflammation, and Drug Discovery

The research in this dissertation explores the bioactivity and biosynthesis of lipoxygenase-derived products from polyunsaturated fatty acids as well as drug discovery. The diseases that cause most of the mortality today are due to chronic inflammation, which is caused by the lack of a proper resolution phase. The pro-resolution molecules are oxylipins catalyzed by oxygenases and derived from fish oil. Dietary, genetic, and environmental effects have a role in altering these lipid signaling pathways and causing inflammation. This thesis examines how these molecules are made, their effects on resolving inflammation, and possible relations to disease. The first chapter investigates human 12-lipoxygenase metabolites from omega-6 docosapentaenoic acid as a promising novel anti-platelet for the treatment of the life-threatening complications of cardiovascular diseases. The second chapter quantifies oxyipins and cytokines in human blood as possible biomarkers for heart failure. The next chapter characterizes the Pseudomonas Aeruginosa lipoxygenase, reveals possible roles in biofilm formation and manipulation of host immune response, and discovers first in class selective inhibitors. Finally, the last chapter describes the resolution of inflammation and the biosynthesis of a class of specialized proresolving mediators, the maresins

Supplementation with omega‐3 or omega‐6 fatty acids attenuates platelet reactivity in postmenopausal women

Postmenopausal women are at increased risk for a cardiovascular event due to platelet hyperactivity. There is evidence suggesting that ω-3 polyunsaturated fatty acids (PUFAs) and ω-6 PUFAs have cardioprotective effects in these women. However, a mechanistic understanding of how these fatty acids regulate platelet function is unknown. In this study, we supplemented postmenopausal women with fish oil (ω-3 fatty acids) or evening primrose oil (ω-6 fatty acids) and investigated the effects on their platelet activity. The effects of fatty acid supplementation on platelet aggregation, dense granule secretion, and activation of integrin αIIbβ3 at basal levels and in response to agonist were tested in postmenopausal women following a supplementation and washout period. Supplementation with fish oil or primrose oil attenuated the thrombin receptor PAR4-induced platelet aggregation. Supplementation with ω-3 or ω-6 fatty acids decreased platelet dense granule secretion and attenuated basal levels of integrin αIIbβ3 activation. Interestingly, after the washout period following supplementation with primrose oil, platelet aggregation was similarly attenuated. Additionally, for either treatment, the observed protective effects post-supplementation on platelet dense granule secretion and basal levels of integrin activation were sustained after the washout period, suggesting a long-term shift in platelet reactivity due to fatty acid supplementation. These findings begin to elucidate the underlying mechanistic effects of ω-3 and ω-6 fatty acids on platelet reactivity in postmenopausal women. Hence, this study supports the beneficial effects of fish oil or primrose oil supplementation as a therapeutic intervention to reduce the risk of thrombotic events in postmenopausal women. https://clinicaltrials.gov/ct2/show/NCT02629497

15-Lipoxygenase-1 biosynthesis of 7S,14S-diHDHA implicates 15-lipoxygenase-2 in biosynthesis of resolvin D5.

The two oxylipins 7S,14S-dihydroxydocosahexaenoic acid (diHDHA) and 7S,17S-diHDHA [resolvin D5 (RvD5)] have been found in macrophages and infectious inflammatory exudates and are believed to function as specialized pro-resolving mediators (SPMs). Their biosynthesis is thought to proceed through sequential oxidations of DHA by lipoxygenase (LOX) enzymes, specifically, by human 5-LOX (h5-LOX) first to 7(S)-hydroxy-4Z,8E,10Z,13Z,16Z,19Z-DHA (7S-HDHA), followed by human platelet 12-LOX (h12-LOX) to form 7(S),14(S)-dihydroxy-4Z,8E,10Z,12E,16Z,19Z-DHA (7S,14S-diHDHA) or human reticulocyte 15-LOX-1 (h15-LOX-1) to form RvD5. In this work, we determined that oxidation of 7(S)-hydroperoxy-4Z,8E,10Z,13Z,16Z,19Z-DHA to 7S,14S-diHDHA is performed with similar kinetics by either h12-LOX or h15-LOX-1. The oxidation at C14 of DHA by h12-LOX was expected, but the noncanonical reaction of h15-LOX-1 to make over 80% 7S,14S-diHDHA was larger than expected. Results of computer modeling suggested that the alcohol on C7 of 7S-HDHA hydrogen bonds with the backbone carbonyl of Ile399, forcing the hydrogen abstraction from C12 to oxygenate on C14 but not C17. This result raised questions regarding the synthesis of RvD5. Strikingly, we found that h15-LOX-2 oxygenates 7S-HDHA almost exclusively at C17, forming RvD5 with faster kinetics than does h15-LOX-1. The presence of h15-LOX-2 in neutrophils and macrophages suggests that it may have a greater role in biosynthesizing SPMs than previously thought. We also determined that the reactions of h5-LOX with 14(S)-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-DHA and 17(S)-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-DHA are kinetically slow compared with DHA, suggesting that these reactions may be minor biosynthetic routes in vivo. Additionally, we show that 7S,14S-diHDHA and RvD5 have anti-aggregation properties with platelets at low micromolar potencies, which could directly regulate clot resolution

Derivation of the Pediatric Acute Gastroenteritis Risk Score to Predict Moderate-to-Severe Acute Gastroenteritis

OBJECTIVES: Although most acute gastroenteritis (AGE) episodes in children rapidly self-resolve, some children go on to experience more significant and prolonged illness. We sought to develop a prognostic score to identify children at risk of experiencing moderate-to-severe disease after an index emergency department (ED) visit. METHODS: Data were collected from a cohort of children 3 to 48 months of age diagnosed with AGE in 16 North American pediatric EDs. Moderate-to-severe AGE was defined as a Modified Vesikari Scale (MVS) score ≥9 during the 14-day post-ED visit. A clinical prognostic model was derived using multivariable logistic regression and converted into a simple risk score. The model’s accuracy was assessed for moderate-to-severe AGE and several secondary outcomes. RESULTS: After their index ED visit, 19% (336/1770) of participants developed moderate-to-severe AGE. Patient age, number of vomiting episodes, dehydration status, prior ED visits, and intravenous rehydration were associated with MVS ≥9 in multivariable regression. Calibration of the prognostic model was strong with a P value of 0.77 by the Hosmer-Lemenshow goodness-of-fit test, and discrimination was moderate with an area under the receiver operator characteristic curve of 0.68 (95% confidence interval [CI] 0.65–0.72). Similarly, the model was shown to have good calibration when fit to the secondary outcomes of subsequent ED revisit, intravenous rehydration, or hospitalization within 72 hours after the index visit. CONCLUSIONS: After external validation, this new risk score may provide clinicians with accurate prognostic insight into the likely disease course of children with AGE, informing disposition decisions, anticipatory guidance, and follow-up care

Pathogen-Specific Effects of Probiotics in Children With Acute Gastroenteritis Seeking Emergency Care: A Randomized Trial

BACKGROUND: It is unknown if probiotics exert pathogen-specific effects in children with diarrhea secondary to acute gastroenteritis. METHODS: Analysis of patient-level data from 2 multicenter randomized, placebo controlled trials conducted in pediatric emergency departments in Canada and the United States. Participants were 3-48 months with \u3e3 diarrheal episodes in the preceding 24 hours and were symptomatic for \u3c72 hours and \u3c7 days in the Canadian and US studies, respectively. Participants received either placebo or a probiotic preparation (Canada-Lactobacillus rhamnosus R0011/Lactobacillus helveticus R0052; US-L. rhamnosus GG). The primary outcome was post-intervention moderate-to-severe disease (ie, ≥9 on the Modified Vesikari Scale [MVS] score). RESULTS: Pathogens were identified in specimens from 59.3% of children (928/1565). No pathogen groups were less likely to experience an MVS score ≥9 based on treatment allocation (test for interaction = 0.35). No differences between groups were identified for adenovirus (adjusted relative risk [aRR]: 1.42; 95% confidence interval [CI]: .62, 3.23), norovirus (aRR: 0.98; 95% CI: .56, 1.74), rotavirus (aRR: 0.86; 95% CI: .43, 1.71) or bacteria (aRR: 1.19; 95% CI: .41, 3.43). At pathogen-group and among individual pathogens there were no differences in diarrhea duration or the total number of diarrheal stools between treatment groups, regardless of intervention allocation or among probiotic sub-groups. Among adenovirus-infected children, those administered the L. rhamnosus R0011/L. helveticus R0052 product experienced fewer diarrheal episodes (aRR: 0.65; 95% CI: .47, .90). CONCLUSIONS: Neither probiotic product resulted in less severe disease compared to placebo across a range of the most common etiologic pathogens. The preponderance of evidence does not support the notion that there are pathogen specific benefits associated with probiotic use in children with acute gastroenteritis. CLINICAL TRIALS REGISTRATION: NCT01773967 and NCT01853124

Association Between Diarrhea Duration and Severity and Probiotic Efficacy in Children With Acute Gastroenteritis.

IntroductionIt is unclear whether the alleged efficacy of probiotics in childhood acute gastroenteritis depends on the duration and severity of symptoms before treatment.MethodsPreplanned secondary analysis of 2 randomized placebo-controlled trials in children 3-48 months of age was conducted in 16 emergency departments in North America evaluating the efficacy of 2 probiotic products (Lactobacillus rhamnosus GG and a combination probiotic: L. rhamnosus and L. helveticus). Participants were categorized in severity groups according to the duration (<24, 24-<72, and ≥72 hours) and the frequency of diarrhea episodes in the 24 hours (≤3, 4-5, and ≥6) before presentation. We used regression models to assess the interaction between pretreatment diarrhea severity groups and treatment arm (probiotic or placebo) in the presence of moderate-to-severe gastroenteritis (Modified Vesikari Scale score ≥9). Secondary outcomes included diarrhea frequency and duration, unscheduled healthcare provider visits, and hospitalization.ResultsA total of 1,770 children were included, and 882 (50%) received a probiotic. The development of moderate-to-severe gastroenteritis symptoms after the initiation of treatment did not differ between groups (probiotic-18.4% [162/882] vs placebo-18.3% [162/888]; risk ratio 1.00; 95% confidence interval 0.87, 1.16; P = 0.95). There was no evidence of interaction between baseline severity and treatment (P = 0.61) for the primary or any of the secondary outcomes: diarrhea duration (P = 0.88), maximum diarrheal episodes in a 24-hour period (P = 0.87), unscheduled healthcare visits (P = 0.21), and hospitalization (P = 0.87).DiscussionIn children 3-48 months with acute gastroenteritis, the lack of effect of probiotics is not explained by the duration of symptoms or frequency of diarrheal episodes before presentation

Association Between Diarrhea Duration and Severity and Probiotic Efficacy in Children With Acute Gastroenteritis

BACKGROUND: It is unclear if the alleged efficacy of probiotics in childhood acute gastroenteritis depends on the duration and severity of symptoms prior to treatment. METHODS: Pre-planned secondary analysis of two randomized placebo-controlled trials in children 3-48 months of age, conducted in sixteen emergency departments in North America evaluating the efficacy of two probiotic products (Lactobacillus rhamnosus GG and a combination probiotic: L. rhamnosus and L. helveticus). Participants were categorized in severity groups according to the duration (< 24h, 24 – < 72h, and ≥ 72h) and the frequency of diarrhea episodes in the 24 hours (≤ 3, 4 – 5 and ≥ 6) prior to presentation. We used regression models to assess the interaction between pre-treatment diarrhea severity groups and treatment arm (probiotic or placebo) on the presence of moderate-to-severe gastroenteritis (Modified Vesikari Scale score ≥9). Secondary outcomes included diarrhea frequency and duration, unscheduled healthcare provider visits and hospitalization. RESULTS: 1,770 children were included, 882 (50%) received a probiotic. The development of moderate-to-severe gastroenteritis symptoms following the initiation of treatment did not differ between groups [probiotic - 18.4% (162/882) vs. placebo - 18.3% (162/888); RR 1.00; 95%CI: 0.87, 1.16; P=0.95]. There was no evidence of interaction between baseline severity and treatment (P=0.61) for the primary or any of the secondary outcomes: diarrhea duration (P=0.88), maximum diarrheal episodes in a 24 hour period (P=0.87), unscheduled health care visits (P=0.21) and hospitalization (P=0.87). CONCLUSION: In children 3 to 48 months with acute gastroenteritis the lack of effect of probiotics is not explained by the duration of symptoms or frequency of diarrheal episodes prior to presentation