Effect of arginine and oscillatory calcium ion (II) on vascular response mediated via nitric oxide signaling in normal and salt sensitive hypertensive rat mesenteric arterioles

Hypertension, a major risk factor in the cardiovascular system, is characterized by an increase in the arterial blood pressure. High dietary sodium is linked to multiple cardiovascular disorders including hypertension. Salt sensitivity, a measure of how the blood pressure responds to salt intake is observed in more than 50% of the hypertension cases. Nitric Oxide (NO), as an endogenous vasodilator serves many important biological roles in the cardiovascular physiology including blood pressure regulation. The physiological concentrations for NO bioactivity are reported to be in 0-500 nM range. Notably, the vascular response to NO is highly regulated within a small concentration spectrum. Hence, much uncertainty surrounds how NO modulates diverse signaling mechanisms to initiate vascular relaxation and alleviate hypertension. Regulating the availability of NO in the vasculature has demonstrated vasoprotective effects. In addition, modulating the NO release by different means has proved to restore endothelial function. In this study we addressed parameters that regulated NO release in the vasculature, in physiology and pathophysiology such as salt sensitive hypertension. We showed that, in the rat mesenteric arterioles, Ca2+ induced rapid relaxation (time constants 20.8 ± 2.2 sec) followed with a much slower constriction after subsequent removal of the stimulus (time constants 104.8 ± 10.0 sec). An interesting observation was that a fourfold increase in the Ca 2+ frequency improved the efficacy of arteriolar relaxation by 61.1%. Our results suggested that, Ca2+ frequency-dependent transient release of NO from the endothelium carried encoded information; which could be translated into different steady state vascular tone. Further, Agmatine, a metabolite of L-arginine, as a ligand, was observed to relax the mesenteric arterioles. These relaxations were NO-dependent and occurred via &agr;-2 receptor activity. The observed potency of agmatine (EC50, 138.7 ± 12.1 ± μM; n=22), was 40 fold higher than L-arginine itself (EC50, 18.3 ± 1.3 mM; n = 5). This suggested us to propose alternative parallel mechanism for L-arginine mediated vascular relaxation via arginine decarboxylase activity. In addition, the biomechanics of rat mesentery is important in regulation of vascular tone. We developed 2D finite element models that described the vascular mechanics of rat mesentery. With an inverse estimation approach, we identified the elasticity parameters characterizing alterations in normotensive and hypertensive Dahl rats. Our efforts were towards guiding current studies that optimized cardiovascular intervention and assisted in the development of new therapeutic strategies. These observations may have significant implications towards alternatives to present methods for NO delivery as a therapeutic target. Our work shall prove to be beneficial in assisting the delivery of NO in the vasculature thus minimizing the cardiovascular risk in handling abnormalities, such as hypertension.

Análise imunoistoquímica após terapia fotodinâmica com cloro-alumínio ftalocianina em tecidos gengivais de humanos

Although photodynamic therapy have been used as a useful tool over the past 30 years in oncology, few clinical trials have been conducted in dentistry. Photodynamic therapy (PDT) uses non - toxic photosensitizers and selective which are administered in target cells followed by local application of visible light, producing reactive oxygen species capable of causing cell death by apoptosis or necrosis, injured the local vasculature, and exert important effects on the im mune system. New generations of photosensitizing agents, such as nanoparticulate phthalocyanines, has shown excellent results in antitumor and antibacterial activity . In this context, the present work constitutes the first clinical protocol of local appli cation of nanoemulsion chloro - aluminum phthalocyanine (AlClFc) followed by irradiation in human gingiva, and analyzed descriptively and comparatively , by means of immunohistochemistry , the expression of RANK , RANKL , OPG and VEGF in a split - mouth model . Eight healthy volunteers with clinical indication for extraction were included in the study . Seven days before the extraction, was injected in the gingiva of participants, 5 μ M of nanoemulsion AlClFc followed by irra diation with diode laser (660nm , 7 J/cm2 ), the contralateral side was used as control. Tissue specimens were removed seven days after the TFD is performed. Tissues sample were divided into two groups (test and con trol groups) for histological and immunohistochemical analysis. Patients were monitored at days, 0, 7, 14 and 30 to assess adverse effects of the therapy. Vascular alterations were seen in gingival samples that received PDT. Areas of edema and vascular con gestion, and intense vascularization were viewed . Additionally, dystrophic calcification in subepithelial region were observed in the test group. The results showed a similar pattern of immunostaining scores of RANK, RANKL and VEGF between the test and co ntrol groups, with no statistically significant difference (p = 0.317, p = 0.777, p = 0 .814, respectively). RANK and RANKL exhibited weak or absent immunostaining in most specimens analyzed. There was n o immunostaining for OPG. VEGF showed moderate to stro ng immunostaining in specimens from the test group. In addition, the clinical study showed that therapy was well tolerated by all patients. Adverse effects were short - time and completely reversible. Taken together, the results presented in this study showe d that PDT mediated by nanoemulsion containing AlClPc is safe for clinical application in gingival tissue and suggests that a strong immunostaining for VEGF after therapy .

Estudo imunoistoquímico do CD34 e podoplanina na doença periodontal

Angiogenesis and lymphangiogenesis are changes that occur due to gingival inflammation caused by microorganisms present in the biofilm, as well as the migration of immune cells and secretion of mediators in the aggressed site. This study aimed to research angiogenesis and lymphangiogenesis in 90 specimens of clinically healthy, with gingivitis and chronic periodontitis gingival tissue biopsies. The histological sections were evaluated by hematoxylin and eosin and the immunohistochemical technique through immunostaining for CD34 and podoplanin. To evaluate the angiogenic and lymphangiogenic indexes we performed a microvessel counting technique. The results showed that there is a correlation between the indexes (p = 0.030), however, we observed that periodontitis showed less lymphatic vessels than clinically healthy gingival tissue (p = 0.016). Podoplanin showed positive staining in the basal layers of the epithelium, and we observed a relationship between immunostaining intensity and the intensity of inflammatory infiltrate, with more intense staining in the presence of severe inflammatory infiltrate (p = 0.033). For this study, we concluded that there are fewer blood vessels in periodontitis compared with clinically healthy gingiva. The signaling present in the inflammatory process and the actual role of gingival blood and lymphatic vasculature are not fully understood, with further studies on angiogenesis and lymphangiogenesis being suggested.

Estudo imunoistoquímico do CD34 e podoplanina na doença periodontal

Angiogenesis and lymphangiogenesis are changes that occur due to gingival inflammation caused by microorganisms present in the biofilm, as well as the migration of immune cells and secretion of mediators in the aggressed site. This study aimed to research angiogenesis and lymphangiogenesis in 90 specimens of clinically healthy, with gingivitis and chronic periodontitis gingival tissue biopsies. The histological sections were evaluated by hematoxylin and eosin and the immunohistochemical technique through immunostaining for CD34 and podoplanin. To evaluate the angiogenic and lymphangiogenic indexes we performed a microvessel counting technique. The results showed that there is a correlation between the indexes (p = 0.030), however, we observed that periodontitis showed less lymphatic vessels than clinically healthy gingival tissue (p = 0.016). Podoplanin showed positive staining in the basal layers of the epithelium, and we observed a relationship between immunostaining intensity and the intensity of inflammatory infiltrate, with more intense staining in the presence of severe inflammatory infiltrate (p = 0.033). For this study, we concluded that there are fewer blood vessels in periodontitis compared with clinically healthy gingiva. The signaling present in the inflammatory process and the actual role of gingival blood and lymphatic vasculature are not fully understood, with further studies on angiogenesis and lymphangiogenesis being suggested.

Preeclampsia : an accelerator–brake defect disorder

Life's perfect partnership starts with the placenta. If we get this right, we have the best chance of healthy life. In preeclampsia, we have a failing placenta. Preeclampsia kills one pregnant woman every minute and the life expectancy of those who survive is greatly reduced. Preeclampsia is treated roughly the same way it was when Thomas Edison was making the first silent movie. Globally, millions of women risk death to give birth each year and almost 300,000 lose their lives in this process. Over half a million babies around the world die each year as a consequence of preeclampsia. Despite decades of research, we lack pharmacological agents to treat it. Maternal endothelial dysfunction is a central phenomenon responsible for the clinical signs of preeclampsia. In the late nineties, we discovered that vascular endothelial growth factor (VEGF) stimulated nitric oxide release. This led us to suggest that preeclampsia arises due to the loss of VEGF activity, possibly due to a rise in soluble Flt-1 (sFlt-1), the natural antagonist of VEGF. Researchers have shown that high sFlt-1 elicits preeclampsia-like signs in pregnant rats and sFlt-1 increases before the clinical signs of preeclampsia in pregnant women. We demonstrated that removing or reducing this culprit protein from preeclamptic placenta restored the angiogenic balance. Heme oxygenase-1 (HO-1 or Hmox1) that generates carbon monoxide (CO), biliverdin (rapidly converted to bilirubin) and iron is cytoprotective. We showed that the Hmox1/CO pathway prevents human placental injury caused by pro-inflammatory cytokines and suppresses sFlt-1 and soluble endoglin release, factors responsible for preeclampsia phenotypes. The other key enzyme we identified is the hydrogen sulfide generating cystathionine-gamma-lyase (CSE or Cth). These are the only two enzyme systems shown to suppress sFlt-1 and to act as protective pathways against preeclampsia phenotypes in animal models. We also showed that when hydrogen sulfide restores placental vasculature, it also improves lagging fetal growth. These molecules act as the inhibitor systems in pregnancy and when they fail, this triggers preeclampsia. Discovering that statins induce these enzymes led us to an RCT to develop a low-cost therapy (StAmP Trial) to prevent or treat preeclampsia. If you think of pregnancy as a car then preeclampsia is an accelerator–brake defect disorder. Inflammation, oxidative stress and an imbalance in the angiogenic milieu fuel the ‘accelerator’. It is the failure in the braking systems (the endogenous protective pathway) that results in the ‘accelerator’ going out of control until the system crashes, manifesting itself as preeclampsia.

Cystathionine γ-lyase regulates arteriogenesis through NO-dependent monocyte recruitment

AIMS: Hydrogen sulfide (H2S) is a vasoactive gasotransmitter that is endogenously produced in the vasculature by the enzyme cystathionine γ-lyase (CSE). However, the importance of CSE activity and local H2S generation for ischaemic vascular remodelling remains completely unknown. In this study, we examine the hypothesis that CSE critically regulates ischaemic vascular remodelling involving H2S-dependent mononuclear cell regulation of arteriogenesis. METHODS AND RESULTS: Arteriogenesis including mature vessel density, collateral formation, blood flow, and SPY angiographic blush rate were determined in wild-type (WT) and CSE knockout (KO) mice at different time points following femoral artery ligation (FAL). The role of endogenous H2S in regulation of IL-16 expression and subsequent recruitment of monocytes, and expression of VEGF and bFGF in ischaemic tissues, were determined along with endothelial progenitor cell (CD34/Flk1) formation and function. FAL of WT mice significantly increased CSE activity, expression and endogenous H2S generation in ischaemic tissues, and monocyte infiltration, which was absent in CSE-deficient mice. Treatment of CSE KO mice with the polysulfide donor diallyl trisulfide restored ischaemic vascular remodelling, monocyte infiltration, and cytokine expression. Importantly, exogenous H2S therapy restored nitric oxide (NO) bioavailability in CSE KO mice that was responsible for monocyte recruitment and arteriogenesis. CONCLUSION: Endogenous CSE/H2S regulates ischaemic vascular remodelling mediated during hind limb ischaemia through NO-dependent monocyte recruitment and cytokine induction revealing a previously unknown mechanism of arteriogenesis.

Advanced Techniques for Image Quality Assessment of Modern X-ray Computed Tomography Systems

X-ray computed tomography (CT) imaging constitutes one of the most widely used diagnostic tools in radiology today with nearly 85 million CT examinations performed in the U.S in 2011. CT imparts a relatively high amount of radiation dose to the patient compared to other x-ray imaging modalities and as a result of this fact, coupled with its popularity, CT is currently the single largest source of medical radiation exposure to the U.S. population. For this reason, there is a critical need to optimize CT examinations such that the dose is minimized while the quality of the CT images is not degraded. This optimization can be difficult to achieve due to the relationship between dose and image quality. All things being held equal, reducing the dose degrades image quality and can impact the diagnostic value of the CT examination.

A recent push from the medical and scientific community towards using lower doses has spawned new dose reduction technologies such as automatic exposure control (i.e., tube current modulation) and iterative reconstruction algorithms. In theory, these technologies could allow for scanning at reduced doses while maintaining the image quality of the exam at an acceptable level. Therefore, there is a scientific need to establish the dose reduction potential of these new technologies in an objective and rigorous manner. Establishing these dose reduction potentials requires precise and clinically relevant metrics of CT image quality, as well as practical and efficient methodologies to measure such metrics on real CT systems. The currently established methodologies for assessing CT image quality are not appropriate to assess modern CT scanners that have implemented those aforementioned dose reduction technologies.

Thus the purpose of this doctoral project was to develop, assess, and implement new phantoms, image quality metrics, analysis techniques, and modeling tools that are appropriate for image quality assessment of modern clinical CT systems. The project developed image quality assessment methods in the context of three distinct paradigms, (a) uniform phantoms, (b) textured phantoms, and (c) clinical images.

The work in this dissertation used the “task-based” definition of image quality. That is, image quality was broadly defined as the effectiveness by which an image can be used for its intended task. Under this definition, any assessment of image quality requires three components: (1) A well defined imaging task (e.g., detection of subtle lesions), (2) an “observer” to perform the task (e.g., a radiologists or a detection algorithm), and (3) a way to measure the observer’s performance in completing the task at hand (e.g., detection sensitivity/specificity).

First, this task-based image quality paradigm was implemented using a novel multi-sized phantom platform (with uniform background) developed specifically to assess modern CT systems (Mercury Phantom, v3.0, Duke University). A comprehensive evaluation was performed on a state-of-the-art CT system (SOMATOM Definition Force, Siemens Healthcare) in terms of noise, resolution, and detectability as a function of patient size, dose, tube energy (i.e., kVp), automatic exposure control, and reconstruction algorithm (i.e., Filtered Back-Projection– FPB vs Advanced Modeled Iterative Reconstruction– ADMIRE). A mathematical observer model (i.e., computer detection algorithm) was implemented and used as the basis of image quality comparisons. It was found that image quality increased with increasing dose and decreasing phantom size. The CT system exhibited nonlinear noise and resolution properties, especially at very low-doses, large phantom sizes, and for low-contrast objects. Objective image quality metrics generally increased with increasing dose and ADMIRE strength, and with decreasing phantom size. The ADMIRE algorithm could offer comparable image quality at reduced doses or improved image quality at the same dose (increase in detectability index by up to 163% depending on iterative strength). The use of automatic exposure control resulted in more consistent image quality with changing phantom size.

Based on those results, the dose reduction potential of ADMIRE was further assessed specifically for the task of detecting small (<=6 mm) low-contrast (<=20 HU) lesions. A new low-contrast detectability phantom (with uniform background) was designed and fabricated using a multi-material 3D printer. The phantom was imaged at multiple dose levels and images were reconstructed with FBP and ADMIRE. Human perception experiments were performed to measure the detection accuracy from FBP and ADMIRE images. It was found that ADMIRE had equivalent performance to FBP at 56% less dose.

Using the same image data as the previous study, a number of different mathematical observer models were implemented to assess which models would result in image quality metrics that best correlated with human detection performance. The models included naïve simple metrics of image quality such as contrast-to-noise ratio (CNR) and more sophisticated observer models such as the non-prewhitening matched filter observer model family and the channelized Hotelling observer model family. It was found that non-prewhitening matched filter observers and the channelized Hotelling observers both correlated strongly with human performance. Conversely, CNR was found to not correlate strongly with human performance, especially when comparing different reconstruction algorithms.

The uniform background phantoms used in the previous studies provided a good first-order approximation of image quality. However, due to their simplicity and due to the complexity of iterative reconstruction algorithms, it is possible that such phantoms are not fully adequate to assess the clinical impact of iterative algorithms because patient images obviously do not have smooth uniform backgrounds. To test this hypothesis, two textured phantoms (classified as gross texture and fine texture) and a uniform phantom of similar size were built and imaged on a SOMATOM Flash scanner (Siemens Healthcare). Images were reconstructed using FBP and a Sinogram Affirmed Iterative Reconstruction (SAFIRE). Using an image subtraction technique, quantum noise was measured in all images of each phantom. It was found that in FBP, the noise was independent of the background (textured vs uniform). However, for SAFIRE, noise increased by up to 44% in the textured phantoms compared to the uniform phantom. As a result, the noise reduction from SAFIRE was found to be up to 66% in the uniform phantom but as low as 29% in the textured phantoms. Based on this result, it clear that further investigation was needed into to understand the impact that background texture has on image quality when iterative reconstruction algorithms are used.

To further investigate this phenomenon with more realistic textures, two anthropomorphic textured phantoms were designed to mimic lung vasculature and fatty soft tissue texture. The phantoms (along with a corresponding uniform phantom) were fabricated with a multi-material 3D printer and imaged on the SOMATOM Flash scanner. Scans were repeated a total of 50 times in order to get ensemble statistics of the noise. A novel method of estimating the noise power spectrum (NPS) from irregularly shaped ROIs was developed. It was found that SAFIRE images had highly locally non-stationary noise patterns with pixels near edges having higher noise than pixels in more uniform regions. Compared to FBP, SAFIRE images had 60% less noise on average in uniform regions for edge pixels, noise was between 20% higher and 40% lower. The noise texture (i.e., NPS) was also highly dependent on the background texture for SAFIRE. Therefore, it was concluded that quantum noise properties in the uniform phantoms are not representative of those in patients for iterative reconstruction algorithms and texture should be considered when assessing image quality of iterative algorithms.

The move beyond just assessing noise properties in textured phantoms towards assessing detectability, a series of new phantoms were designed specifically to measure low-contrast detectability in the presence of background texture. The textures used were optimized to match the texture in the liver regions actual patient CT images using a genetic algorithm. The so called “Clustured Lumpy Background” texture synthesis framework was used to generate the modeled texture. Three textured phantoms and a corresponding uniform phantom were fabricated with a multi-material 3D printer and imaged on the SOMATOM Flash scanner. Images were reconstructed with FBP and SAFIRE and analyzed using a multi-slice channelized Hotelling observer to measure detectability and the dose reduction potential of SAFIRE based on the uniform and textured phantoms. It was found that at the same dose, the improvement in detectability from SAFIRE (compared to FBP) was higher when measured in a uniform phantom compared to textured phantoms.

The final trajectory of this project aimed at developing methods to mathematically model lesions, as a means to help assess image quality directly from patient images. The mathematical modeling framework is first presented. The models describe a lesion’s morphology in terms of size, shape, contrast, and edge profile as an analytical equation. The models can be voxelized and inserted into patient images to create so-called “hybrid” images. These hybrid images can then be used to assess detectability or estimability with the advantage that the ground truth of the lesion morphology and location is known exactly. Based on this framework, a series of liver lesions, lung nodules, and kidney stones were modeled based on images of real lesions. The lesion models were virtually inserted into patient images to create a database of hybrid images to go along with the original database of real lesion images. ROI images from each database were assessed by radiologists in a blinded fashion to determine the realism of the hybrid images. It was found that the radiologists could not readily distinguish between real and virtual lesion images (area under the ROC curve was 0.55). This study provided evidence that the proposed mathematical lesion modeling framework could produce reasonably realistic lesion images.

Based on that result, two studies were conducted which demonstrated the utility of the lesion models. The first study used the modeling framework as a measurement tool to determine how dose and reconstruction algorithm affected the quantitative analysis of liver lesions, lung nodules, and renal stones in terms of their size, shape, attenuation, edge profile, and texture features. The same database of real lesion images used in the previous study was used for this study. That database contained images of the same patient at 2 dose levels (50% and 100%) along with 3 reconstruction algorithms from a GE 750HD CT system (GE Healthcare). The algorithms in question were FBP, Adaptive Statistical Iterative Reconstruction (ASiR), and Model-Based Iterative Reconstruction (MBIR). A total of 23 quantitative features were extracted from the lesions under each condition. It was found that both dose and reconstruction algorithm had a statistically significant effect on the feature measurements. In particular, radiation dose affected five, three, and four of the 23 features (related to lesion size, conspicuity, and pixel-value distribution) for liver lesions, lung nodules, and renal stones, respectively. MBIR significantly affected 9, 11, and 15 of the 23 features (including size, attenuation, and texture features) for liver lesions, lung nodules, and renal stones, respectively. Lesion texture was not significantly affected by radiation dose.

The second study demonstrating the utility of the lesion modeling framework focused on assessing detectability of very low-contrast liver lesions in abdominal imaging. Specifically, detectability was assessed as a function of dose and reconstruction algorithm. As part of a parallel clinical trial, images from 21 patients were collected at 6 dose levels per patient on a SOMATOM Flash scanner. Subtle liver lesion models (contrast = -15 HU) were inserted into the raw projection data from the patient scans. The projections were then reconstructed with FBP and SAFIRE (strength 5). Also, lesion-less images were reconstructed. Noise, contrast, CNR, and detectability index of an observer model (non-prewhitening matched filter) were assessed. It was found that SAFIRE reduced noise by 52%, reduced contrast by 12%, increased CNR by 87%. and increased detectability index by 65% compared to FBP. Further, a 2AFC human perception experiment was performed to assess the dose reduction potential of SAFIRE, which was found to be 22% compared to the standard of care dose.

In conclusion, this dissertation provides to the scientific community a series of new methodologies, phantoms, analysis techniques, and modeling tools that can be used to rigorously assess image quality from modern CT systems. Specifically, methods to properly evaluate iterative reconstruction have been developed and are expected to aid in the safe clinical implementation of dose reduction technologies.

Influence of Outlet Boundary Conditions on Cerebrovascular Aneurysm Hemodynamics

Computational fluid dynamic (CFD) studies of blood flow in cerebrovascular aneurysms have potential to improve patient treatment planning by enabling clinicians and engineers to model patient-specific geometries and compute predictors and risks prior to neurovascular intervention. However, the use of patient-specific computational models in clinical settings is unfeasible due to their complexity, computationally intensive and time-consuming nature. An important factor contributing to this challenge is the choice of outlet boundary conditions, which often involves a trade-off between physiological accuracy, patient-specificity, simplicity and speed. In this study, we analyze how resistance and impedance outlet boundary conditions affect blood flow velocities, wall shear stresses and pressure distributions in a patient-specific model of a cerebrovascular aneurysm. We also use geometrical manipulation techniques to obtain a model of the patient’s vasculature prior to aneurysm development, and study how forces and stresses may have been involved in the initiation of aneurysm growth. Our CFD results show that the nature of the prescribed outlet boundary conditions is not as important as the relative distributions of blood flow through each outlet branch. As long as the appropriate parameters are chosen to keep these flow distributions consistent with physiology, resistance boundary conditions, which are simpler, easier to use and more practical than their impedance counterparts, are sufficient to study aneurysm pathophysiology, since they predict very similar wall shear stresses, time-averaged wall shear stresses, time-averaged pressures, and blood flow patterns and velocities. The only situations where the use of impedance boundary conditions should be prioritized is if pressure waveforms are being analyzed, or if local pressure distributions are being evaluated at specific time points, especially at peak systole, where the use of resistance boundary conditions leads to unnaturally large pressure pulses. In addition, we show that in this specific patient, the region of the blood vessel where the neck of the aneurysm developed was subject to abnormally high wall shear stresses, and that regions surrounding blebs on the aneurysmal surface were subject to low, oscillatory wall shear stresses. Computational models using resistance outlet boundary conditions may be suitable to study patient-specific aneurysm progression in a clinical setting, although several other challenges must be addressed before these tools can be applied clinically.

ROLE OF STAT1 AND CXCL10 IN THE TUMOUR IMMUNE MICROENVIRONMENT OF HIGH-GRADE SEROUS OVARIAN CANCER

High-grade serous ovarian cancer (HGSC) is the most prevalent epithelial ovarian cancer characterized by late detection, metastasis and resistance to chemotherapy. Previous studies on the tumour immune microenvironment in HGSC identified STAT1 and CXCL10 as the most differentially expressed genes between treatment naïve chemotherapy resistant and sensitive tumours. Interferon-induced STAT1 is a transcription factor, which induces many genes including tumour suppressor genes and those involved in recruitment of immune cells to the tumour immune microenvironment (TME), including CXCL10. CXCL10 is a chemokine that recruits tumour infiltrating lymphocytes (TILs) and exhibits angiostatic function. The current study was performed to determine the effects of differential STAT1 and CXCL10 expression on HGSC disease progression and TME. STAT1 expression and intratumoural CD8+ T cells were evaluated as prognostic and predictive biomarkers via immunohistochemistry on 734 HGSC tumours accrued from the Terry Fox Research Institute-Canadian Ovarian Experimental Unified Resource. The combined effect of STAT1 expression and CD8+ TIL density was confirmed as prognostic and predictive companion biomarkers in the second independent biomarker validation study. Significant positive correlation between STAT1 expression and intratumoral CD8+ TIL density was observed. The effects of enforced CXCL10 expression on HGSC tumour growth, vasculature and immune tumour microenvironment were studied in the ID8 mouse ovarian cancer cell engraftment in immunocompetent C57BL/6 mice. Significant decrease in tumour progression in mice injected with ID8 CXCL10 overexpressing cells compared to mice injected with ID8 vector control cells was observed. Multiplexed cytokine analysis of ascites showed differential expression of IL-6, VEGF and CXCL9 between the two groups. Endothelial cell marker staining showed differences in tumour vasculature between the two groups. Immune transcriptomic profiling identified distinct expression profiles in genes associated with cytokines, chemokines, interferons, T cell function and apoptosis between the two groups. These findings provide evidence that STAT1 is an independent biomarker and in combination with CD8+ TIL density could be applied as novel immune-based biomarkers in HGSC. These results provide the basis for future studies aimed at understanding mechanisms underlying differential tumour STAT1 and CXCL10 expression and its role in pre-existing tumour immunologic diversity, thus potentially contributing to biomarker guided immune modulatory therapies.

THE IMPORTANCE OF MHC-INDEPENDENT NATURAL KILLER CELL RECEPTORS IN THE IMMUNOREGULATION OF MURINE PREGNANCY

Numerous leukocyte populations are essential for pregnancy success. Uterine natural killer (uNK) cells are chief amongst these leukocytes and represent a unique lineage with limited cytotoxicity but abundant angiokine production. They possess a distinct phenotype of activating and inhibitory receptors that recognize major histocompatibility complex (MHC) molecules, such as the killer immunoglobulin like receptors (KIRs; mouse Ly49), and MHC-independent activating receptors, including the aryl hydrocarbon receptor (AHR) and natural cytotoxicity receptor 1 (NCR1). While the roles of MHC-dependent receptors are widely addressed in pregnancy, MHC-independent receptors are relatively unstudied. This thesis investigated the roles of MHC-independent receptors in promotion of mouse pregnancy and characterized early leukocyte interactions in the presence and absence of NCR1. It was hypothesized that loss of MHC-independent receptors impairs uNK cell development resulting in aberrations in leukocyte function and decidual vasculature. Implantation sites from Ahr-/- and Ncr1Gfp/Gfp mice were assessed using whole mount in situ immunohistochemistry (WM-IHC) and histochemical techniques. Leukocyte interactions identified during preliminary WM-IHC studies were confirmed as immune synapses. The novel identification of immune synapses in early mouse pregnancy compelled further examination of leukocyte conjugates in wildtype C57BL/6 and Ncr1Gfp/Gfp mice. In Ahr-/- and Ncr1Gfp/Gfp mice, receptor loss resulted in reduced uNK cell diameters, impaired decidual vasculature, and failures in spiral artery remodeling. Ahr-/- mice had severe fertility deficits whereas Ncr1Gfp/Gfp mice had increased fetal resorption indicating differing receptor requirements in pregnancy success. NCR1 loss primarily affected uNK cell maturation and function as identified by alterations in granule ultrastructure, lytic protein expression, and angiokine production. Leukocyte conjugates were frequent in early C57BL/6 decidua basalis and included uNK cells conjugating first with antigen presenting cells and then with T cells. Overall conjugate formation was reduced in the absence of NCR1, but specific uNK cell conjugations were unaffected by receptor loss. While KIR-MHC interactions are associated with numerous pregnancy complications in humans, the role of other uNK cell receptors are not well characterized. These results illustrate the importance of MHC-independent receptors in uNK cell activation during early pregnancy in mice and encourage further studies of pregnancy complications that may occur independently of maternal KIR-MHC contributions.

IMMUNE-ANGIOGENESIS MECHANISMS ASSOCIATED WITH PORCINE PREGNANCY SUCCESS AND FAILURE

Spontaneous fetal loss (25-40%) leading to decrease in litter size is a significant concern to the pork industry. A deficit in the placental vasculature has emerged as one of the important factors associated with fetal loss. During early pig pregnancy, the endometrium becomes enriched with immune cells recruited by conceptus-derived signals including specific chemokine stimuli. These immune cells assist in various aspects of placental development and angiogenesis. Recent evidence suggests that microRNAs (miRNAs: small non-coding RNAs that regulate gene expression) regulate immune cell development and their functions. In addition, intercellular communication including exchange of biomolecules (e.g. miRNAs) between the conceptus and endometrium regulate key developmental processes during pregnancy. To understand the biological significance of immune cell enrichment, regulation of their functions by miRNAs and transfer of miRNAs across the maternal fetal-interface, we screened specific sets of chemokines and pro- and anti-angiogenic miRNAs in endometrial lymphocytes (ENDO LY), endometrium, and chorioallantoic membrane (CAM) isolated from conceptus attachment sites (CAS) during early, gestation day (gd)20 and mid-pregnancy (gd50). We report increased expression of selected chemokines including CXCR3 and CCR5 in ENDO LY and CXCL10, CXCR3, CCL5, CCR5 in endometrium associated with arresting CAS at gd20. Some of these differences were also noted at the protein level (CXCL10, CXCR3, CCL5, and CCR5) in endometrium and CAM. We report for the first time significant differences for miRNAs involved in immune cell-derived angiogenesis (miR-296-5P, miR-150, miR-17P-5P, miR-18a, and miR-19a) between ENDO LY associated with healthy and arresting CAS. Significant differences were also found in endometrium and CAM for some miRNAs (miR-17-5P, miR-18a, miR-15b-5P, and miR-222). Finally, we confirm that placenta specific-exosomes contain proteins and 14 select miRNAs including miR-126-5P, miR-296-5P, miR-16, and miR-17-5P that are of relevance to early implantation events. We further demonstrated the bidirectional exosome shuttling between porcine trophectoderm cells (PTr2) and porcine aortic endothelial cells (PAOEC). PTr2-derived exosomes were able to modulate the endothelial cell proliferation that is crucial for the establishment of pregnancy. Our data unravels the selected chemokines and miRNAs associated with immune cell-regulated angiogenesis and reconfirm that exosome mediated cell-cell communication opens-up new avenues to understand porcine pregnancy.

The restorative role of annexin A1 at the blood–brain barrier

Annexin A1 is a potent anti-inflammatory molecule that has been extensively studied in the peripheral immune system, but has not as yet been exploited as a therapeutic target/agent. In the last decade, we have undertaken the study of this molecule in the central nervous system (CNS), focusing particularly on the primary interface between the peripheral body and CNS: the blood–brain barrier. In this review, we provide an overview of the role of this molecule in the brain, with a particular emphasis on its functions in the endothelium of the blood–brain barrier, and the protective actions the molecule may exert in neuroinflammatory, neurovascular and metabolic disease. We focus on the possible new therapeutic avenues opened up by an increased understanding of the role of annexin A1 in the CNS vasculature, and its potential for repairing blood–brain barrier damage in disease and aging.

Effects of menthol on rat tail artery mediated by TRPM8 and voltage-gated calcium channels

Transient receptor potential melastatin 8 (TRPM8) is the principal cold and menthol receptor channel. Characterized primarily for its cold sensing role in sensory neurons, it is expressed and functional in several non-neuronal tissues, including vasculature. We previously demonstrated that menthol causes vasoconstriction and vasodilatation in isolated arteries, depending on vascular tone. Here we investigated calcium's role in responses mediated by TRPM8 ligands in rat tail artery myocytes using patch-clamp electrophysiology and ratiometric Ca2+ recording. Isometric contraction studies examined actions of TRPM8 ligands in the presence/absence of L-type calcium channel blocker. Menthol (300 μM), a concentration typically used to induce TRPM8 currents, strongly inhibited L-type voltage-dependent Ca2+ current (L-ICa) in myocytes, especially it's sustained component, most relevant for depolarisation-induced vasoconstriction. In contraction studies, with nifedipine present (10 μM) to abolish L-ICa contribution to phenylephrine (PE)-induced vasoconstrictions of vascular rings, a marked increase in tone was observed with menthol. Menthol-induced increases in PE-induced vasoconstrictions were mediated predominantly by Ca2+-release from sarcoplasmic reticulum, since they were significantly inhibited by cyclopiazonic acid. Pre-incubation of vascular rings with a TRPM8 antagonist strongly inhibited menthol-induced increases in PE-induced vasoconstrictions, thus confirming specific role of TRPM8. Finally, two other common TRPM8 agonists, WS-12 and icilin, inhibited L-ICa. Thus, TRPM8 channels are functionally active in rat tail artery myocytes and play a distinct direct stimulatory role in control of vascular tone. However, indirect effects of TRPM8 agonists, which are unrelated to TRPM8, are mediated by inhibition of L-type Ca2+ channels, and largely obscure TRPM8-mediated vasoconstriction.

The Influence of Age, Cardiorespiratory Fitness, Exercise and Sedentary Behaviours on Circulating Angiogenic Cells and Cell Surface Receptor Expression.

Cardiovascular disease (CVD) is the biggest killer of people in western civilisation. Age is a significant risk factor for the development for CVD, and treatments and therapies to address this increased risk are crucial to quality of life and longevity. Exercise is one such intervention which has been shown to reduce CVD risk. Age is also associated with endothelial dysfunction, reduced angiogenic capabilities, and reduced ability to repair the vessel wall. Circulating angiogenic cells (CACs) are a subset of circulating cells which assist in the repair and growth of the vasculature and in the maintenance of endothelial function. Reductions in these cells are observed in those with vascular disease compared to age-matched healthy controls. Exercise may reduce CVD risk by improvements in number and/or function of these CACs. Data was collected from human volunteers of various ages, cardiorespiratory fitness (CRF) levels and latent viral infection history status to investigate the effects of chronological age, CRF, viral serology and other lifestyle factors, such as sedentary behaviours and exercise on CACs. The levels of CACs in these volunteers were measured using four colour flow cytometry using various monoclonal antibodies specific to cell surface markers that are used to identify specific subsets of these CACs. In addition, the response to acute exercise of a specific subset of these CACs, termed ‘angiogenic T-cells’ (TANG) were investigated, in a group of well-trained males aged 20-40 years, using a strenuous submaximal exercise bout. Advancing age was associated with a decline in various subsets of CACs, including bone marrow-derived CD34+ progenitors, putative endothelial progenitor cells (EPCs) and also TANG cells. Individuals with a higher CRF were more likely to have higher circulating numbers of TANG cells, particularly in the CD4+ subset. CRF did not appear to modulate CD34+ progenitors or EPC subsets. Increasing sitting time was associated with reduction in TANG cells, but after correcting for the effects of fitness, sitting time no longer negatively affected the circulating number of these cells. Acute exercise was a powerful stimulus for increasing the number of TANG cells (140% increase), potentially through an SDF-1:CXCR4-dependent mechanism, but more studies are required to investigate this. Latent CMV infection was associated with higher number of TANG cells (CD8+), but only in 18-40 year old individuals, and not in an older age group (41-65 year old). The significance of this has yet to be understood. In conclusion, advancing age may contribute to increased CVD risk partly due to the observed reductions in angiogenic cells circulating in the peripheral compartment. Maintaining a high CRF may attenuate this CVD reduction by modulating TANG cell number, but potentially not CD34+ progenitor or EPC subsets. Acute exercise may offer a short window for vascular adaptation through the mobilisation of TANG cells into the circulation.

Neutrophil Chemotaxis in Multiple Chemoattractant Gradients

Chemotaxis, the phenomenon in which cells move in response to extracellular chemical gradients, plays a prominent role in the mammalian immune response. During this process, a number of chemical signals, called chemoattractants, are produced at or proximal to sites of infection and diffuse into the surrounding tissue. Immune cells sense these chemoattractants and move in the direction where their concentration is greatest, thereby locating the source of attractants and their associated targets. Leading the assault against new infections is a specialized class of leukocytes (white blood cells) known as neutrophils, which normally circulate in the bloodstream. Upon activation, these cells emigrate out of the vasculature and navigate through interstitial tissues toward target sites. There they phagocytose bacteria and release a number of proteases and reactive oxygen intermediates with antimicrobial activity. Neutrophils recruited by infected tissue in vivo are likely confronted by complex chemical environments consisting of a number of different chemoattractant species. These signals may include end target chemicals produced in the vicinity of the infectious agents, and endogenous chemicals released by local host tissues during the inflammatory response. To successfully locate their pathogenic targets within these chemically diverse and heterogeneous settings, activated neutrophils must be capable of distinguishing between the different signals and employing some sort of logic to prioritize among them. This ability to simultaneously process and interpret mulitple signals is thought to be essential for efficient navigation of the cells to target areas. In particular, aberrant cell signaling and defects in this functionality are known to contribute to medical conditions such as chronic inflammation, asthma and rheumatoid arthritis. To elucidate the biomolecular mechanisms underlying the neutrophil response to different chemoattractants, a number of efforts have been made toward understanding how cells respond to different combinations of chemicals. Most notably, recent investigations have shown that in the presence of both end target and endogenous chemoattractant variants, the cells migrate preferentially toward the former type, even in very low relative concentrations of the latter. Interestingly, however, when the cells are exposed to two different endogenous chemical species, they exhibit a combinatorial response in which distant sources are favored over proximal sources. Some additional results also suggest that cells located between two endogenous chemoattractant sources will respond to the vectorial sum of the combined gradients. In the long run, this peculiar behavior could result in oscillatory cell trajectories between the two sources. To further explore the significance of these and other observations, particularly in the context of physiological conditions, we introduce in this work a simplified phenomenological model of neutrophil chemotaxis. In particular, this model incorporates a trait commonly known as directional persistence - the tendency for migrating neutrophils to continue moving in the same direction (much like momentum) - while also accounting for the dose-response characteristics of cells to different chemical species. Simulations based on this model suggest that the efficiency of cell migration in complex chemical environments depends significantly on the degree of directional persistence. In particular, with appropriate values for this parameter, cells can improve their odds of locating end targets by drifting through a network of attractant sources in a loosely-guided fashion. This corroborates the prediction that neutrophils randomly migrate from one chemoattractant source to the next while searching for their end targets. These cells may thus use persistence as a general mechanism to avoid being trapped near sources of endogenous chemoattractants - the mathematical analogue of local maxima in a global optimization problem. Moreover, this general foraging strategy may apply to other biological processes involving multiple signals and long-range navigation.