This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

¡iAbstract¡j Neurogenic inflammation is an acute inflammatory tissue response, that is mediated by sensory axon reflex. Accompanied with neurogenic inflammation, plasma extravasation, occurs in the eyes, esophagus, bladder, joints, the tip of tongue, and the respiratory tract of the mammal. Recently, many studies have investigated the neurogenic inflammation by electrical stimulation of nerves and intravascular injection of irritants. Upon stimulation, the sensory nerve endings in mucosa can release neuropeptides such as substance P, that causes formation of the venular endothelial gaps, plasma extravasation and tissue edema in various organs. Substance P also cause smooth muscle contraction and mucus secretion in the respiratory tract. Neurogenic plasma extravasation has been studied extensively in the trachea, and bronchi, but rarely in the esophagus. It is known that a plexus of substance P-immunoreactive axons exists in the mucosal and submucosal layers. They play an important role in releasing substance P to act on the receptors of the venular endothelium through diffusion. Based on plasma extravasation and other studies related to the respiratory tract, the purpose of the present study was to investigate neurogenic inflammatory response in the esophagus of the digestive tract. In this study, capsaicin (90 µg/ml/kg) and substance P (3 µg/ml/kg) were used as the irritant and inflammatory mediator, respectively to reduce neurogenic inflammation in the esophagus. India ink was used to label the affected venules. The magnitude of the neurogenic inflammation was expressed as area density of India ink-labeled leaky venules. Histopathological changes in the esophageal tissue were studied under the light microscope. The result of this study indicated that capsaicin at the dose of (90 µg/ml/kg) and substance P at the dose of (3 µg/ml/kg) caused similar magnitude of inflammation in the esophagus. India ink-labeled venules distributed like a network in the mucosal tissue and in connective tissue of the submucosal layer. The upper, middle and lower parts of esophagus exhibited the same degree of inflammatory response, that was similar to that in the lower respiratory tract as the previous studies reported. These results suggest that nerve branches from the vagal trunk send sensory axons to innervate both the esophagus and airways.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "August 2007" Includes bibliographical references.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Research into automatic retina image analysis has become increasingly important, not just in ophthalmology but also in other clinical specialities such as cardiology and neurology. In the retina, blood vessels can be directly visualised non-invasively in-vivo, and hence it serves as a "window" to cardiovascular and neurovascular complications. Biomarker research, i.e. investigating associations between the morphology of the retinal vasculature (as a means of revealing microvascular health or disease) and particular conditions affecting the body or brain could play an important role in detecting disease early enough to impact on patient treatment and care. A fundamental requirement of biomarker research is access to large datasets to achieve sufficient power and significance when ascertaining associations between retinal measures and clinical characterisation of disease. Crucially, the vascular changes that appear can affect arteries and veins differently. An essential part of automatic systems for retinal morphology quantification and biomarker extraction is, therefore, a computational method for classifying vessels into arteries and veins. Artery-vein classification enables the efficient extraction of biomarkers such as the Arteriolar to Venular Ratio, which is a well-established predictor of stroke and other cardiovascular events. While structural parameters of the retinal vasculature such as vessels calibre, branching angle, and tortuosity may individually convey some information regarding specific aspects of the health of the retinal vascular network, they do not convey a global summary of the branching pattern and its state or condition. The retinal vascular tree can be considered a fractal structure as it has a branching pattern that exhibits the property of self-similarity. Fractal analysis, therefore, provides an additional means for the quantitative study of changes to the retinal vascular network and may be of use in detecting abnormalities related to retinopathy and systemic diseases. In this thesis, new developments to fully automated retinal vessel classification and fractal analysis were explored in order to extract potential biomarkers. These novel processes were tested and validated on several datasets of retinal images acquired with fundus cameras. The major contributions of this thesis include: 1) developing a fully automated retinal blood vessel classification technique, 2) developing a fractal analysis technique that quantifies regional as well as global branching complexity, 3) validating the methods using multiple datasets, and 4) applying the proposed methods in multiple retinal vasculature analysis studies.

Bladder cancer, as one of the most common cancer types and with high recurrence risk, is considered a candidate for novel immunotherapy strategies. An important aspect of the research for immunotherapy drug development for bladder cancer is to study the tumor microenvironment (TME) and it’s immune contexture. Besides tumor-infiltrating lymphocytes (TILs) as the main drivers of anti-tumor response, recent studies revealed the importance of tumor-associated tertiary lymphoid structures (TLSs) and high endothelial venules (HEVs) in the TME. Structures similar to these were found to spontaneously form in the orthotopic MB49 model used for bladder cancer research in our group. The aim of this study was to perform a deeper characterization of the TME in this model, by using immunofluorescent staining and microscopy. Specifically, the co-localization of tumor infiltrating lymphocytes (CD8+ and CD4+ T cells, CD19+ B cells), CD11c+ dendritic cells and HEVs along with CCL21 signaling were analyzed within orthotopic MB49 tumors, with and without immune stimulation. The quantification of cells expressing CD8, CD19 and CD11c immune markers, CCL21 levels, vascular density and numbers of HEVs, showed higher densities within the immune-stimulated tumors, indicating a rapid effect of immune stimulation on increasing immune cell infiltration and vascular density after only 24 hours post CpG therapy. Also, the highest frequency of TILs, CCL21 chemokine and vascular density was located in regions of the tumor border indicating that these regions should be studied further in depth as a potential target for entry of cells to the tumor with immunotherapy or as a model of the tumor microenvironment since tumor cell density is maintained high in these locations.

Sickle cell disease (SCD) is a severe genetic disease, affecting over 100,000 in the United States and millions worldwide. Individuals suffer from stroke, acute chest syndrome, and cardiovascular complications. Much of these associated morbidities are primarily mediated by blockages of the microvasculature, events termed vaso-occlusive crises (VOCs). Despite its prevalence and severity, the pathophysiological mechanisms behind VOCs are not well understood, and novel experimental tools and methods are needed to further this understanding. Microfluidics and computational fluid dynamics (CFD) are rapidly growing fields within biomedical research that allow for inexpensive simulation of the in vivo microenvironment prior to animal or clinical trials. This study includes the development of a CFD model capable of simulating diseased and healthy blood flow within a series of microfluidic channels. Results will be utilized to further improve the development of microfluidic systems.

Estudos demonstram que o treinamento aeróbio é capaz de reduzir a razão parede/luz (RP/L) de arteríolas musculares esqueléticas e reduzir a pressão arterial média, PAM (em hipertensos), além de causar bradicardia de repouso e aumentar a densidade de capilares e vênulas (em hipertensos e normotensos) após 13 semanas de treinamento. Investigamos em ratos normotensos (WKY, Wistar Kyoto) e hipertensos espontâneos (SHR) as alterações estruturais da microcirculação muscular esquelética, cardíaca e renal, relacionando-as aos valores de PAM e frequência cardíaca (FC) em diferentes fases de um protocolo de treinamento. Para tal, WKY e SHR (2 meses de idade) foram submetidos ao protocolo de treinamento físico de baixa intensidade por tempos crescentes (semanas 0, 1, 2, 4, 8 e 12) ou mantiveram-se sedentários (semanas 0 e 12). Ao final de cada tempo de estudo foram mensurados de modo direto a PAM e a FC e coletados coração, rim e músculos temporal, sóleo e gastrocnêmio. Realizou-se análise da RP/L e quantificação de capilares e vênulas (através do ácido periódico de Schiff). O treinamento aeróbio aumentou a densidade capilar e venular de WKY e SHR (apenas em territórios exercitados e com maior magnitude nos hipertensos), em seguida causou redução da razão parede/luz das arteríolas musculares esqueléticas (apenas em SHR, precocemente e em maior magnitude em territórios exercitados) e só então reduziu a PAM de SHR e a FC de WKY e SHR. A partir dos resultados obtidos, podemos dizer que alterações estruturais da microcirculação antecedem a melhora dos níveis pressóricos de hipertensos e ainda, são capazes de proporcionar melhoras vasculares aos normotensos / Previous observations have shown that aerobic training reduce the wall/lumen ratio (RW/L) of skeletal muscle arterioles and reduce mean arterial pressure (MAP) in hypertensive rats, cause bradycardia, and increase capillary and venular density also in hypertensive and normotensive rats after 13 weeks of training. We investigated simultaneously the time-course changes of arterioles remodeling, MAP, HR and capillary and venular density during the development of low-intensity exercise protocol. Normotensive rats (WKY, Wistar Kyoto) and spontaneously hypertensive (SHR), two-months old were submitted to aerobic training protocol (weeks 0, 1, 2, 4, 8 and 12) or remained sedentary (weeks 0 and 12). In each study time were measured the MAP and HR and collected heart, kidney, temporalis, soleus and gastrocnemius muscles to analyze the RW/L and quantificate capillaries and venules (by Periodic Acid-Schiff staining). Aerobic training increased capillary and venular density of WKY and SHR (only in exercised territories), caused a reduction of the RW/L of skeletal muscle arterioles (only in SHR, early and in greater magnitude in exercised territories) and only then reduced MAP of SHR and HR of WKY and SHR. The structural changes of microcirculation preceded improvement of blood pressure levels in hypertensive rats and provided vascular improvements to normotensive rats

Estudos demonstram que o treinamento aeróbio é capaz de reduzir a razão parede/luz (RP/L) de arteríolas musculares esqueléticas e reduzir a pressão arterial média, PAM (em hipertensos), além de causar bradicardia de repouso e aumentar a densidade de capilares e vênulas (em hipertensos e normotensos) após 13 semanas de treinamento. Investigamos em ratos normotensos (WKY, Wistar Kyoto) e hipertensos espontâneos (SHR) as alterações estruturais da microcirculação muscular esquelética, cardíaca e renal, relacionando-as aos valores de PAM e frequência cardíaca (FC) em diferentes fases de um protocolo de treinamento. Para tal, WKY e SHR (2 meses de idade) foram submetidos ao protocolo de treinamento físico de baixa intensidade por tempos crescentes (semanas 0, 1, 2, 4, 8 e 12) ou mantiveram-se sedentários (semanas 0 e 12). Ao final de cada tempo de estudo foram mensurados de modo direto a PAM e a FC e coletados coração, rim e músculos temporal, sóleo e gastrocnêmio. Realizou-se análise da RP/L e quantificação de capilares e vênulas (através do ácido periódico de Schiff). O treinamento aeróbio aumentou a densidade capilar e venular de WKY e SHR (apenas em territórios exercitados e com maior magnitude nos hipertensos), em seguida causou redução da razão parede/luz das arteríolas musculares esqueléticas (apenas em SHR, precocemente e em maior magnitude em territórios exercitados) e só então reduziu a PAM de SHR e a FC de WKY e SHR. A partir dos resultados obtidos, podemos dizer que alterações estruturais da microcirculação antecedem a melhora dos níveis pressóricos de hipertensos e ainda, são capazes de proporcionar melhoras vasculares aos normotensos / Previous observations have shown that aerobic training reduce the wall/lumen ratio (RW/L) of skeletal muscle arterioles and reduce mean arterial pressure (MAP) in hypertensive rats, cause bradycardia, and increase capillary and venular density also in hypertensive and normotensive rats after 13 weeks of training. We investigated simultaneously the time-course changes of arterioles remodeling, MAP, HR and capillary and venular density during the development of low-intensity exercise protocol. Normotensive rats (WKY, Wistar Kyoto) and spontaneously hypertensive (SHR), two-months old were submitted to aerobic training protocol (weeks 0, 1, 2, 4, 8 and 12) or remained sedentary (weeks 0 and 12). In each study time were measured the MAP and HR and collected heart, kidney, temporalis, soleus and gastrocnemius muscles to analyze the RW/L and quantificate capillaries and venules (by Periodic Acid-Schiff staining). Aerobic training increased capillary and venular density of WKY and SHR (only in exercised territories), caused a reduction of the RW/L of skeletal muscle arterioles (only in SHR, early and in greater magnitude in exercised territories) and only then reduced MAP of SHR and HR of WKY and SHR. The structural changes of microcirculation preceded improvement of blood pressure levels in hypertensive rats and provided vascular improvements to normotensive rats

Abstract The function of the transmembrane protein FasL is to complex with the Fas receptor in a target cell and induce target cell apoptosis. Fas/FasL-mediated apoptosis plays important role in immunoregulation. FasL expression is mostly seen in activated lymphocytes. We have characterized endothelial FasL expression in different functional compartments of lymph nodes and gut-associated lymphoid tissue. Furthermore, we have explored the functional role of endothelial FasL expression by analyzing correlation with apoptosis of lymphocyte subpopulations in lymph nodes and by assessing endothelial expression under different conditions by activation of immune functions in gastrointestinal mucosa. Immunohistochemical stainings (Fas, FasL, CD3, CD20, CD19, CD23, CD56, FVIII) were performed on 20 reactive lymph node tissues (I and II), 60 pediatric endoscopy biopsy samples (III) or 60 samples from gut resections (IV). A double-staining method combining apoptosis detection with the TUNEL-method and lymphocyte classification with FasL, Fas and cell lineage markers was optimized. Patient groups included non-pathological lymph nodes, pediatric cow’s milk-sensitive enteropathy, pediatric celiac disease, appendicitis, ulcerative colitis and Crohn’s disease. Control groups included normal biopsy samples from pediatric patients and non-pathological resecate samples from the appendix, colon or ileum to correspond to patient groups. Quantitative analysis (positive vessels or cells per mm2) was performed thoroughly for each anatomical region. In a subset of patients, soluble FasL in the serum was quantified with standard enzyme-linked immunosorbent assay. In reactive lymph nodes FasL expression was predominantly present in high endothelial venules located in the paracortical area, where apoptotic T and B lymphocytes, some expressing Fas, were subsequently found. In the gut wall vascular FasL expression was seen in high endothelial vessels near lymphoid follicles. Serum FasL was elevated in children with an abundance of mucosal lymphoid follicles. In IBD, vascular FasL was upregulated in ulcers and in the submucosa of colons affected by Crohn’s disease. The results indicate that endothelial FasL is characteristically present in high endothelial venules of lymphoid tissues. Detection of apoptotic Fas expressing lymphocytes adjacent to such vessels supports the idea that endothelial FasL functions as a selective gatekeeper by inducing apoptosis of Fas+ lymphocytes entering from the blood stream. / Tiivistelmä Solukalvon läpäisevän proteiinin, FasL:n, tehtävä on sitoutua kohdesolun Fas-reseptoriin ja indusoida kohdesolun apoptoosi. Fas/FasL-välitteinen apoptoosi on merkittävä tekijä immunologisessa säätelyssä. FasL ilmentyy pääsääntöisesti aktivoituneissa lymfosyyteissä. Olemme kuvanneet tutkimuksessamme FasL:n endoteelistä ilmentymistä imukudoksen eri toiminnallisissa alueissa ja suoliston lymfaattisessa kudoksessa. Lisäksi kartoitimme endoteelin FasL:n toiminnallista merkitystä analysoimalla sen yhteyttä lymfosyyttien alaryhmien apoptoosiin imusolmukkeissa ja arvioimalla FasL:n endoteelistä ilmentymistä suoliston limakalvon immunologisesti erilaisissa sairauksissa. Teimme immunohistokemiallisia värjäyksiä (Fas, FasL, CD3, CD20, CD19, CD23, CD56 ja FVIII) 20 reaktiiviselle imusolmukkeelle (I ja II), 60 lapsen endoskooppiselle biopsianäytteelle (III) sekä 60 suoliresekaattinäytteelle (IV). Optimoimme kaksoisvärjäysmenetelmän, missä yhdistettiin apoptoosin havainnointimenetelmä TUNEL ja FasL-, Fas- tai solulinjamarkkeri. Potilasryhmiin kuului potilaita, joilla oli normaalit imusolmukkeet, sekä potilaita, jotka sairastivat lasten viivästynyttä lehmänmaitoallergiaa, lasten keliakiaa, umpilisäketulehdusta, haavaista paksusuolitulehdusta tai Crohnin tautia. Verrokkiryhmiin kuului normaaleja biopsianäytteitä lapsipotilailta sekä terveitä resekaattinäytteitä umpilisäkkeestä sekä paksu- tai sykkyräsuolesta potilasryhmien mukaisesti. Jokaiselle anatomiselle alueelle suoritimme perusteellisen määrällisen analyysin (positiivista suonta tai solua per mm2). Osalle ryhmistä suoritimme seerumin liukoisen FasL:n määrityksen entsyymivälitteisellä immunosorbenttimäärityksellä. Reaktiivisissa imusolmukkeissa FasL:n ilmentyminen näkyi pääsääntöisesti parakortikaalialueen korkeaendoteelisissä venuleissa, missä myös apoptoottiset T- ja B-lymfosyytit (joista osa ilmensi Fasia) sittemmin näkyivät. Suoliston seinämässä havaitsimme verisuoniperäistä FasL:n ilmentymistä korkeaendoteelisissä suonissa lymfaattisten itukeskusten lähettyvillä. Niillä lapsipotilailla, joilla havaitsimme limakalvon lymfaattisten itukeskuksien lisääntymistä, oli myös seerumin FasL-pitoisuus koholla. Tulehduksellisissa suolistosairauksissa verisuoniperäinen FasL oli lisääntynyt limakalvon haavaumissa sekä Crohnin tautia sairastavien potilaiden submukoosassa. Tulokset osoittavat verisuoniperäisen FasL:n tyypillisesti ilmentyvän imukudoksen korkeaendoteelisissa suonissa. Apoptoosin havaitseminen Fasia ilmentävissä lymfosyyteissä näiden suonien läheisyydessä tukee ajatusta siitä, kuinka verisuoniperäinen FasL toimii valikoivana portinvartijana ja aiheuttaa Fas-positiivisten lymfosyyttien apoptoosin estämällä niiden pääsyn verenkierrosta.