Long term consumption of a high fat diet (HFD) increases the risk of developing Metabolic Syndrome and type 2 diabetes. This led us to hypothesize that long term HFD consumption impairs immune tolerance to the intestinal bacteria. Our studies had two goals. First, we characterized the effect of long term HFD consumption on the systemic immune response by comparing C57BL6 mice fed a HFD and low fat diet (LFD). Plasma immunoglobulin G (IgG) against Escherichia coli (LF-82), E. coli (Nissle 1917), Bacteroides thetaiotaomicron and Lactobacillus acidophilus were measured by a lab-developed ELISA. Fasting blood glucose and inflammation were measured in LFD mice and HFD mice. To test whether our findings were clinically relevant, anti-bacterial IgG and TNF-α were measured in plasma samples from lean healthy individuals, obese non-diabetics and obese diabetics. Our second aim was to investigate the relationship between HFD consumption and intestinal immunity. The effect of HFD consumption on immune responses in the GI tract was assessed by measuring fecal IgA levels in HFD mice and LFD mice. HFD mice had higher plasma IgG against the LF82 strain of Escherichia coli as well as higher plasma TNF-α, neutrophil percentage and fasting blood glucose levels. Obese diabetics had higher plasma IgG against the LF82 strain of E. coli than lean healthy controls. Studies on the effect of HFD on intestinal immunity revealed that HFD mice had lower fecal IgA than LFD mice. Our findings are novel in that they show an association between long term HFD consumption, systemic inflammatory immune responses to pathogenic intestinal bacteria and insulin resistance. These studies also showed that HFD consumption may impair intestinal immunity.

Summary of the work Abdominal aortic aneurysm (AAA) is a chronic degenerative disease that usually affects men over 65 years with an estimated prevalence of 5%. Aneurysm rupture represents a catastrophic event which carries a mortality rate of almost 90%. Current therapeutic options for AAAs measuring 5.5 cm in diameter or larger are based on prophylactic surgery, including conventional open reconstruction and endovascular stent-graft insertion. For patients with small asymptomatic AAAs (4.0 up to 5.5 cm in diameter), evidence from two recent large randomized controlled trials indicates no long-term survival benefit from immediate elective surgical repair as compared to imaging surveillance until aneurysm expands to 5.5 cm. This highlights the need for development of novel medical management strategies, including selective pharmacologic approaches, directed at preventing aneurysm expansion. In this regard, it is expected that a detailed knowledge of the pathobiology of human AAA lesion and a better understanding of pathophysiological mechanisms underlying initiation and progression of aneurysmal degeneration, particularly the specific involvement of T lymphocytes, will have special relevance to this challenging issue. Inflammatory and helper T-cell responses in abdominal aortic aneurysm : controversial issues Innate and inflammatory responses to endovascular versus open AAA repair. The occurrence of early acute systemic inflammatory responses after conventional open AAA repair is widely recognized and is thought to lead to the development of organ dysfunction and multiple organ failure, responsible for a large proportion of morbidity and mortality associated with aortic surgery. New therapeutic strategies designed to avoid ischemia-reperfusion injury related to aortic cross-clamping and to minimize the degree of tissue damage have thus been developed recently. Specifically, the advent of endovascular techniques has radically extended management options for patients with AAA. Although the method is believed to offer a clear short-term benefit over open repair, notably as regards restricted perioperative haemodynamic parameter fluctuations, reduced blood loss, briefer duration of surgery, shorter hospital stay, and lower 30-day mortality and complication rates, conflicting data are available regarding the exact nature and extent of the inflammatory events arising after such endoluminal procedures ; while several authors have indeed reported that endovascular AAA repair can determine a less intense and extensive inflammatory response, others have unexpectedly observed that the method may elicit a strong inflammatory response, the so-called « postimplantation syndrome ». Adaptive cellular immune responses in human aneurysmal aortic lesion. The inflammatory nature of AAA disease has long been suggested by the presence of a great number of CD4+ T lymphocytes in the outer media and adventitia of human AAA lesion. Interestingly, such infiltrating T-cell populations may have significant implications in the process of aneurysm dilation, since cytokines produced by T cells, notably IFN-gamma, have previously been shown to modulate production of matrix-degrading enzymes by resident macrophages and to induce apoptosis of medial SMCs. Through these key pathological mechanisms, T cells could potentially contribute to orchestrate aortic wall connective tissue disordered remodeling and degradation, and promote extensive disruption of elastic media, ultimately leading to aneurysmal degeneration. Nevertheless, despite their relative abundance in human AAA wall tissues, there is limited and controversial information as regards the functional profile of lesional lymphocytes, the exact nature of aortic wall adaptive cellular responses, and the etiologic role of T cells and their cytokines in initiation and progression of the aneurysmal process. Indeed, both Th1-type and Th2-type responses have been identified in human studies and experimental animal models of AAA. Aims of the work The main objectives of our work were to explore the innate and adaptive cellular immune responses in human AAA. In the first part of our work, we aimed to examine prospectively innate and inflammatory responses arising in a non-randomised cohort of patients undergoing endovascular versus open AAA repair. In the second part of our work, we focused our efforts on characterizing the nature of adaptive cellular immune responses and the phenotypic and functional repertoire of T cells in human AAA wall tissues obtained from a consecutive series of patients undergoing open AAA repair. Specifically, we sought to determine whether type 1 or type 2 responses occur predominantly in advanced AAA lesion. Main experimental findings Limited inflammatory response after endovascular AAA repair. Serial peripheral venous blood samples were collected preoperatively, immediately after declamping or insertion of endograft, and after 1, 3, 6, 12, 24, 48, and 72 hours. We first examined the acute phase reaction and liberation of complement cascade products using turbidimetric method and nephelometry. We found that endovascular repair produced lower postoperative CRP, leucocytosis, neutrophilia, and C3d/C3 ratio as compared to open surgery. We next analyzed surface expression of activation markers on peripheral CD3+ T cells using flow cytometry. We observed a strong upregulation of CD38 after open but not endovascular repair. Analysis of CD69 and CD25 molecules revealed no perioperative fluctuations in any group. We then investigated release of various circulating soluble cell adhesion molecules, proinflammatory cytokines, and chemokines using enzyme-linked immunosorbent assays. We demonstrated that both procedures are characterized by similar increases in ICAM-1 and IL-6 levels. Finally, tendency towards high levels of TNF-alpha and IL-8 was detected in endovascular repair, but data failed to reach statistical significance. Predominance of type 1 CD4+ T cells in human aneurysmal aortic lesion. We have developed a tissue enzymatic digestion and cell extraction procedure to isolate intact mononuclear cells from aortic wall segments. This original cell isolation protocol enabled us to examine ex vivo the presence, phenotype, and cytokine secretion profile of infiltrating T lymphocytes freshly isolated from human AAA tissues for comparison with their circulating counterparts using flow cytometry. We found that both populations of infiltrating CD4+ and CD8+ T cells display a unique activated memory phenotype, as assessed by an increased expression of CD69 and HLA-DR activation antigens, downregulation of CD62L molecule, and predominant expression of the CD45RO isoform characteristics of memory cells. In addition, we identified the presence in human aneurysmal aortic wall lesion of CD4+ T cells producing high levels of IFN-gamma but not IL-4, reflecting their type 1 nature. In an additional series of experiments, cytokine gene expression was determined in whole aneurysmal and non-diseased aortic samples using LightCycler-based quantitative real-time reverse transcription-polymerase chain reaction. The molecular basis of type 1 or type 2 dominant responses was further specified by analyzing mRNA levels of transcription factors specifically involved in Th1 or Th2 differentiation such as T-bet and GATA-3. We demonstrated that aneurysmal aortic specimens exhibit high transcript levels of IFN-gamma but not IL-4, and consistently overexpressed the IFN-g-promoting cytokine IL-12 and the type 1-restricted transcription factor T-bet, further establishing the prominent type 1 nature of aortic wall responses. Moreover, such selective tissue expression of IL-12 and T-bet in the vessel microenvironment points to a potential role for these signals in directing aortic wall responses towards a type 1 phenotype. Conclusions Our findings indicate that endovascular AAA repair is associated with a lesser degree of acute phase reaction, peripheral T-cell activation, and release of complement proteins as compared to conventional open surgery, suggesting that the innate and inflammatory responses to AAA repair are significantly attenuated by the endovascular approach as compared to the traditional open reconstruction. These results support the view that the endoluminal procedure represents an attractive alternative to open surgery for the treatment of large aneurysms. On the other hand, we have demonstrated that Th1 cell infiltrates predominate in human end-stage AAA lesion. These observations are relevant for helping clarify the pathobiology of human AAA tissues and defining prospects for the prevention of aneurysm expansion. Indeed, identification of such infiltrating populations of IFN-gamma-producing CD4+ T cells not only provide new insights into the pathogenesis of the disorder, but could also serve as a basis for the development of novel medical management strategies directed at preventing aneurysm formation and progression, including therapeutic approaches based on the modulation of aortic wall responses and designed to selectively target T-cell activation and cytokine production. In this respect, the present work provides experimental evidence in support of the emerging concept that, although multifactorial, aneurysm disease may be regarded as a Th1-driven immunopathological condition, and suggests that strategies targeting IFN-gamma could be a particularly exciting and fruitful avenue for further investigation. Ongoing clinical and basic research in these areas can be expected to yield design of promising pharmacologic approaches to control AAA expansion. From a clinical perspective, such efforts have the potential to dramatically influence both the outcome and management of this common and life threatening condition.

Injury, infection and tissue malfunction are triggers of inflammation which if not regulated may acquire new characteristics that result in pathological outcomes. Since innate immunity plays a key role in the resolution of acute inflammation knowledge of the regulation of this component of the host response is relevant to understanding processes in disease progression and therefore has potential clinical benefits. In this thesis I have applied zebrafish as a model organism to investigate the response of innate immune cells to qualitatively distinct inflammatory signals in the absence of adaptive immunity. Using a zebrafish embryo wound injury model I have investigated leukocyte migration profiles by in vivo imaging. In response to wound alone leukocytes migrated to the site of injury with predominantly random walk behaviour. However, the addition of lipopolysaccharide (LPS) enhanced recruitment and influenced the directionality of leukocyte migration to the wound. I demonstrate that leukocyte dynamic behaviour is also dependent on the location of the cells. The LPS enhanced directionality and reduced the random walk behaviour of the leukocytes, and these effects were ablated in the presence of the p38 mitogenactivated protein kinase (MAPK) specific inhibitor SB203580. Cytokine gene profiling in adult zebrafish leukocytes reveals that LPS can stimulate a pro-inflammatory response via the activation of p38 MAPK characteristic of mammalian innate immune responses. It is documented in mammalian innate immune cells that LPS can modulate Notch mediated signalling and thereby cell function. Using zebrafish with null mutations in Notch, which provide an unbiased in vivo model, I have investigated the influence of Notch signalling on leukocyte recruitment and demonstrate that migration to a wound injury is reduced. However, this effect is due to decreased cell numbers and not altered function as the Notch signalling inhibitor DAPT had no effect of recruitment to wound injury. The defect in myelomonocyte numbers was also present in adult zebrafish and this was partially compensated for by an increase in lymphocytes. The experimental results that I report here highlight zebrafish as a model 2 organism for studying the function and regulation of innate immunity. The unique optical translucency, which permits in vivo imaging of host responses in real-time, facilitates the analysis of the innate immune response to different inflammatory signals and immune modulators.

Currently, there is a growing body of research characterizing the blood-brain barrier (BBB) under normal physiological conditions; however, little is known about BBB regulation under pathophysiological conditions, such as inflammatory pain. This dissertation elucidates peripheral inflammatory pain effects on the BBB both functionally in terms of permeability and structurally via tight junction (TJ) protein expression and regulation.Inflammation was produced by subcutaneous injection of formalin, lambda-carrageenan, or complete Freund's adjuvant (CFA) into the right hind paw of rats. In situ perfusion and Western blot analyses were performed to assess BBB integrity after inflammatory insult. In situ brain perfusion determined that peripheral inflammation significantly increased the uptake of a membrane impermeant marker, sucrose into the cerebral hemispheres in all inflammatory models. Subsequently, a 0-168h time course study of lambda-carrageenan-induced inflammatory pain elicited a biphasic increase in BBB permeability of sucrose with the first phase occurring from 1-6h and the second phase occuring at 48h. Lambda-carrageenan-induced inflammatory pain also increased brain uptake of a commonly used analgesic, codeine at the same time-points. This is the first known observation that peripheral inflammation results in greater analgesic drug uptake to the brain. This uptake also correlated with its antinociceptive profile over a 168h time course. This suggests the presence of inflammatory pain may be an important consideration in therapeutic drug dosing, potential adverse effects and/or neurotoxicity.Western blot analyses showed altered TJ protein expression during peripheral inflammation. Occludin significantly decreased in the lambda-carrageenan- and CFA-treated groups. ZO-1 expression was significantly increased in all pain models. Claudin-1 protein expression was present at the BBB and remained unchanged during inflammation. Actin expression was significantly increased in the lambda-carrageenan- and CFA-treated groups. Over a 72h time period with lambda-carrageenan-induced inflammatory pain, altered TJ protein expression of occludin and ZO-1 correlated with permeability changes in BBB function. This is the first report of peripheral inflammation inducing alterations in TJs and increasing permeability of the BBB. This dissertation demonstrates that changes in the structure of TJs leading to alterations in the BBB may have important clinical ramifications concerning central nervous system homeostasis and therapeutic drug delivery.

Obesity-related diseases account for the majority of morbidity and mortality in U.S. adults. An estimated 4 million women in the United States deliver an infant annually, of which approximately 34% are overweight/obese prior to pregnancy. More than 30% of these women gain weight that exceeds the IOM’s recommendations; increasing their risk of postpartum weight retention and possibly increasing their risk of greater weight gain and retention over time. This research sought to test the efficacy of a traditional MED diet for 4-months on weight loss/control and biomarkers of inflammation in breastfeeding women compared to women randomized to the USDA’s MyPyramid diet for Pregnancy and Breastfeeding (control diet). At baseline, the women (N=129) were 29.7±4.6 years, overweight (BMI: 27.2±4.9 kg/m2), and primarily non-Hispanic white (75.2%). The majority of women were exclusively breastfeeding (73.6%) and a mean 17.5 weeks postpartum. Adherence to the MED diet was evaluated via calculation of the MED diet score from validated FFQs administered pre- and post- the diet intervention. Anthropometric measurements (body weight, body fat, and waist and hip circumference) and biosamples (blood, urine, and breast milk) were collected at baseline and 4-months (diet completion). Biomarkers of inflammation (IL-6 and TNF-α) were assessed via standard ELISA kits. The MED diet score was increased by 0.68±2.74 and 0.27±1.57 for the MED and control group, respectively. Increases in fish and dairy intake and a decrease in meat/poultry intake were significantly different between diet groups (P<0.05). Participants in both diet groups demonstrated significant (P=0.002) reductions in all anthropometric measurements; no significant between group differences were shown. A significant decrease in TNF-α, but not IL-6, was demonstrated in both diet groups. There were no significant between group differences. Both the MED diet and the USDA’s MyPyramid diet were effective in reducing anthropometric measurements and inflammation in postpartum breastfeeding women.

No description available.

No description available.

The study evaluated whether the activity of the indoleamine 2,3 dioxygenase (IDO) enzyme is increased post-stroke and contributes to the development of post-stroke depression (PSD) via tryptophan (TRP) depletion and neurotoxic kynurenine (KYN) metabolite production. The activity of IDO was measured using the KYN/TRP ratio. Participants were assessed for depression severity using the Center for Epidemiological Studies Depression Scale (CES-D). Blood TRP, KYN, large neutral amino acids and cytokines were measured and compared. Fifty-four (mean age=69.9±15.2, male=52.7%, mean NIHSS=7.3±4.6) patients within 28.9±40.3 days of stroke were separated into two groups: non-depressed (n=38, CES-D=6.1±4.9) and those with significant depressive symptoms (n=16, CES-D=26.8±10.8). Higher mean KYN/TRP ratios were demonstrated in stroke patients with depressive symptoms (non-depressed=69.3±36.9 vs. depressive symptoms=78.3±42.0, F3,50=4.61, p=0.006) after controlling for LNAA (p=0.026) and hypertension (p=0.039). As the KYN/TRP ratio reflects decreased TRP and increased neurotoxic KYN metabolites, both mechanisms may play an etiological role in PSD.

Cold-water immersion (CWI) is a common recovery modality used to facilitate restoration of pre-exercise muscle force generation and soreness following high-intensity exercise. Although it is commonly used by athletes and commonly studied in sport science, evidence is equivocal regarding its efficacy. We compared 4 CWI protocols (10 or 30 minutes at 10 or 20°C) of different durations and temperatures with passive rest for their effects on drop jump and squat jump height, inflammation (IL-6, IL-10, IL-8, MPO, IL-1β, TNFα, IFNγ, GM-CSF, IL-2), and ratings of soreness/impairment following high-intensity intermittent sprint-exercise. CWI for 10 minutes at 10°C promoted restoration of force generation, while CWI for 30 minutes at 10°C was associated with lower ratings of soreness/impairment, but higher plasma IL-8 and MPO at 2 hours post-exercise. Overall, minor functional benefits of CWI for 10 minutes at 10°C were observed, while longer duration CWI protocols may increase post-exercise inflammation.

The study evaluated whether the activity of the indoleamine 2,3 dioxygenase (IDO) enzyme is increased post-stroke and contributes to the development of post-stroke depression (PSD) via tryptophan (TRP) depletion and neurotoxic kynurenine (KYN) metabolite production. The activity of IDO was measured using the KYN/TRP ratio. Participants were assessed for depression severity using the Center for Epidemiological Studies Depression Scale (CES-D). Blood TRP, KYN, large neutral amino acids and cytokines were measured and compared. Fifty-four (mean age=69.9±15.2, male=52.7%, mean NIHSS=7.3±4.6) patients within 28.9±40.3 days of stroke were separated into two groups: non-depressed (n=38, CES-D=6.1±4.9) and those with significant depressive symptoms (n=16, CES-D=26.8±10.8). Higher mean KYN/TRP ratios were demonstrated in stroke patients with depressive symptoms (non-depressed=69.3±36.9 vs. depressive symptoms=78.3±42.0, F3,50=4.61, p=0.006) after controlling for LNAA (p=0.026) and hypertension (p=0.039). As the KYN/TRP ratio reflects decreased TRP and increased neurotoxic KYN metabolites, both mechanisms may play an etiological role in PSD.