<p>Asthma involves a systemic element that includes the mobilization and lung-accumulation of bone marrow-derived endothelial progenitor cells (EPC). This traffic may be driven by the stromal cell derived factor-1α (SDF-1α)/CXCR4 axis, where SDF1-α is a potent progenitor cell chemoattractant.</p> <p>Interfering with EPC lung-accumulation by administering AMD3100, a CXCR4 antagonist, was previously shown to be associated with the modulation of airway angiogenesis and airway hyperresponsiveness. However, since eosinophils express CXCR4, it is unknown whether AMD3100 acted directly on EPC or indirectly through its anti-inflammatory effects on eosinophils.</p> <p>We investigated the role that eosinophilic inflammation plays in the lung-homing of EPCs and airway angiogenesis in allergic asthmatic response by utilizing eosinophil deficient (PHIL) mice.</p> <p>Wild-type BALB/c (WT) and PHIL mice underwent a chronic house dust mite (HDM) exposure protocol. Treatment groups were administered AMD3100. Outcome measurements were made 24hrs post final exposure and included: flow cytometry to enumerate lung-extracted EPCs, immunostaining for von Willebrand factor to assess bronchial vascularity, bronchoalveolar lavage for airway inflammation, haematoxylin and eosin stain to enumerate eosinophils, picrosirius red stain to assess collagen deposition, and measurement of airway resistance to increasing intranasal doses of methacholine.</p> <p>HDM exposed mice had a significant increase in EPC lung accumulation, bronchial vascularity, airway inflammation, collagen deposition and airway hyperresponsiveness (AHR) in both WT and PHIL groups, with some indices at lower levels in PHIL mice. Concurrent treatment with AMD3100 significantly attenuated EPC lung homing, bronchial vascularity, eosinophil numbers in lung tissue and AHR, but not collagen deposition in WT mice. AMD3100 treatment significantly attenuated all indices in PHIL mice.</p> <p>The findings of this study show that, EPC-driven angiogenesis and the development of AHR in allergic airway responses are independent of eosinophils, the presence of these cells, however, may have a role in worsening of the pathology of allergic airways disease.</p> / Master of Science (MSc)

In many chronic inflammatory diseases, patients suffer from skeletal muscle weakness, exacerbating their symptoms. Serum levels of tumor necrosis factor-alpha (TNF) and sphingomyelinase are increased, suggesting their possible role in the progression of this weakness. This dissertation focuses on the role that reactive oxygen species (ROS) and nitric oxide (NO) play in mediating TNF-induced skeletal muscle weakness and to what extent sphingolipid signaling mediates cellular response to TNF. The first aim of this work was to identify which endogenous oxidant species stimulated by TNF contributes to skeletal muscle weakness. In C57BL/6 mice (n=38), intraperitoneal injection of TNF elicited a 25% depression of diaphragm contractile function. In separate experiments, diaphragm fiber bundles harvested from mice (n=39) and treated with TNF ex vivo showed a 38% depression of contractile function compared to untreated controls. Using ROS and NO-sensitive fluorescence microscopy in parallel with a genetic knockout animal model, TNF-induced contractile dysfunction was found to be mediated by NO generated by a specific isoform of nitric oxide synthase (NOS), nNOS. Basal levels of ROS were necessary co-mediators, but were not sufficient to elicit TNF-induced diaphragm weakness. The second aim of this dissertation was to investigate the extent to which sphingolipids could serve as a signaling cascade post-TNF stimulus leading to the generation of NO in skeletal muscle. The effects of TNF exposure in C2C12 skeletal muscle cells were studied in vitro using mass spectroscopy to measure sphingolipid metabolism and fluorescent microscopy to quantify oxidant production. TNF exposure was associated with significant mean increases in sphingosine (+52%), general oxidant activity (+33%), and NO production (+14%). These increases were due to specific modulation of nNOS as demonstrated by siRNA knockdown of neutral ceramidase and nNOS, and confirmed by pharmacologic inhibition using N-Oleoylethanolamine and di-methylsphingosine. In summary, these findings confirm NO as a major causative oxidant contributing to TNF’s deleterious phenotype in skeletal muscle. Moreover, the work suggests a new role for sphingosine in skeletal muscle and warrants further study of the enzymatic regulation of sphingosine to advance the discovery of new therapies for patients suffering from chronic inflammation.

There is a chronic lack of donor hearts to meet the need for heart transplant both in the US and worldwide. Further, the use of available hearts is limited by the short period between collection and implantation during which the heart can be safely preserved ex vivo. Using mid-thermic Langendorff machine perfusion, we have been able to preserve the metabolic function of a healthy heart for up to 8 hours, twice the limit for current static cold storage. We have also been able to preserve the metabolic function of a damaged DCD Heart collected 30 minutes after cardiac arrest for a period of 8 hours. We further investigated whether it was possible to improve the preservation of DCD heart using treatment with 10 μM Triiodothyronine to stimulate the tissue metabolism and we did find a reduction in damage markers in the treated DCD hearts as compared to the untreated group.

Com o intuito de avaliar sistema de suspensão mecânica da parede abdominal, elaborou-se protocolo experimental, estudando-se trinta cães, distribuídos em três grupos experimentais de maneira aleatória: grupo pneumoperitônio com gás carbônico, grupo suspensão pela cavidade peritoneal e grupo suspensão abdominal (intraperitoneal) pela tela subcutânea, para documentar alterações metabólicas, hemodinâmicas e respiratórias em cada grupo, compará-las e verificar possíveis vantagens ou desvantagens dos métodos. Os animais foram monitorizados com eletrocardiograma, cateter de Swan-Ganz e pressão arterial invasiva, sendo colhidas amostras de sangue arterial e venoso misto nos momentos basal, 0, 15, 30, 60, 90, 120 minutos e 30 minutos após o término do procedimento, anotando-se para cada momento os parâmetros hemodinâmicos. Calculouse a média e o desvio padrão dos parâmetros, sendo os diferentes grupos comparados pelo teste de Kruskal-Wallis, e nos momentos em que se encontrou diferença estatística entre os três grupos, comparou-se os grupos dois a dois pelo teste de Mann-Whitney. Observou-se, no grupo pneumoperitônio em relação aos grupos suspensão, queda significativa do pH sangüíneo, elevação do bicarbonato, elevação da pressão venosa central e elevação da pressão parcial de gás carbônico, diferenças estas estatisticamente significativas, com p<0,05, não havendo diferenças estatísticas entre os grupos suspensão abdominal e subcutânea / With the purpose of evaluate abdominal wall lifting system, a experimental protocol was elaborated, and thirty dogs, distributed randomly in three experimental groups, pneumoperitoneum with carbon dioxide gas, abdominal (intraperitoneal) suspension and subcutaneous suspension, were studied to document metabolic, hemodynamic and respiratory alterations, compare then and verify if there are some advantage or disadvantage in each method. The animals were monitored with electrocardiogram, Swan-Ganz catheter and invasive arterial blood pressure, and samples of arterial and venous blood were collected at the moments basal, 0, 15, 30, 60, 90 and 120 minutes, and 30 minutes after the end of procedure, registering at each moment the hemodynamical parameters. The three groups were statically compared with Kruskal-Wallis test and, at the moments with statistical differences, the groups were compared two by two with Mann-Whitney test. At the group pneumoperitoneum was observed significant decrease of blood pH, raising of bicarbonate, central venous pressure, and partial carbon dioxide blood pressure in relation to suspension groups, and those differences were statistically significant with p<0.05, without significant differences between abdominal and subcutaneous suspension groups

Heart failure (HF) induced by aortic pressure over-load is associated with increased coronary artery stiffness. Perivascular adipose tissue (PVAT) and advanced glycation end products (AGE) both promote arterial stiffness. However, the mechanisms by which coronary PVAT promotes arterial stiffness and the efficacy of exercise to prevent coronary stiffness are unknown. The present study hypothesized both chronic continuous and interval exercise training would prevent coronary artery stiffness associated with inhibition of PVAT secreted AGE. Yucatan mininature swine were divided into four groups: control-sedentary (CON), aortic-banded sedentary heart failure (HF), aortic-banded HF continuous exercise trained (HF+CONT), and aortic-banded HF interval exercise trained (HF+IT). Coronary artery stiffness was assessed by ex vivo mechanical testing and coronary artery elastin, collagen and AGE-related proteins were assessed by immunohistochemistry. HF promoted coronary artery stiffness with reduced elastin content and greater AGE accumulation which was prevented by chronic continuous and interval exercise training. HF PVAT secreted higher AGE compared with CON and was prevented in the HF+CONT and HF+IT groups. Young healthy mouse aortas cultured in HF PVAT conditioned media had increased stiffness, lower elastin content and AGE accumulation compared with CON, which was prevented by PVAT from the HF+CONT and HF+IT groups. HF coronary PVAT secreted greater interleukin-6 (IL-6) and IL-8 compared to CON which was prevented by both continuous and interval exercise training regimens. We conclude chronic continuous and interval exercise is a potential therapeutic strategy to prevent coronary artery stiffness via inhibition of PVAT-derived AGE secretion in a pre-clinical mini-swine model of pressure overload-induced HF.

Background: Platelet activating factor (PAF) is a phospholipid synthesized by the action of phospholipase A2 and acetyl transferase. PAF possesses a wide range of biological activities. In the lung of the fetus and newborn, PAF binds to its G protein couple receptor to evoke its biological activities via a well-defined signaling pathway. High levels of PAF receptor (PAFr) activity in fetal ovine lung vascular smooth muscle cells (PVSMC) at baseline has previously been demonstrated, a finding that is further perpetuated by conditions of hypoxia similar to fetal lung environment. Additionally in fetal ovine PVSMC, a cross-talk between PAFr-mediated cell signaling and activity of the vasodilator cyclic nucleotides cGMP and cAMP acting via their respective receptors protein kinase (PK) G and PKA has been shown. The interaction of PAF with its receptor has been implicated in the pathogenesis of persistent pulmonary hypertension in the newborn (PPHN) which has a high incidence of hospitalization and death of newborn infants. Successful transition of fetus to newborn life entails a mechanism whereby vasoconstrictors necessary for fetal existence are abrogated in the immediate newborn. Hypothesis: We hypothesize that PPHN results from the failure to down regulate PAFr- mediated activity and /or failure to up-regulate activity of the vasodilators cGMP and cAMP. PPHN is triggered by chronic intrauterine or postnatal hypoxia. Then newborn PVSMC undergo hyperplasia and hypertrophy, which over time, results in irreversible vascular remodeling. Methods: My study aims to employ in vitro models to delineate the consequences of PAF-PAFr mediated pathway in the pharmacological effects of the cAMP-PKA and cGMP-PKG signaling and the involvement of this cross-talk in the pathogenesis of PPHN. I modeled my cell culture studies to mimic the low oxygen environment of fetal lungs (hypoxia), the normal oxygen environment of newborn lungs (normoxia) and high oxygen environment (hyperoxia) to which the newborn lung may be exposed in incidental clinical condition of PPHN. I studied the effect of PAF, a vasoconstrictor, cAMP/cGMP, vasodilators, and other inhibitors of the PAFr pathway on growth of newborn PVSMC, by DNA synthesis, and measured their effects on expression of mitogenic and non-mitogenic proteins. Results: We found that both hypoxia and hyperoxia decreased cell growth even in the presence of PAF which up-regulates cell growth in fetal PVSMC. Also PAF treatment of cells resulted in down regulation of the vasodilator proteins, PKA and PKG. Conclusion: Our data suggests that in the lung of the newborn a high activity of PAF-PAFr mediated activities will worsen the condition of PPHN imposed on the newborn lung by environmental or therapeutic conditions. We can speculate that, in the long run, these findings may translate into the establishment of less toxic protein-based management of PPHN.

[Truncated abstract] Respiratory system impedance (Zrs) is commonly measured at low to medium frequencies (0.5 - 40 Hz) to infer structural and physiological information about the respiratory system. Coupled with the use of mathematical models of the lungs, Zrs has been used to partition the behaviour of the conductive airways and the respiratory tissues. High-frequency (HF) Zrs beyond 100 Hz has been comparatively less studied and understood. Past work has revealed spectral features at high frequencies termed antiresonances, marked by peaks in the real part of Zrs, often coinciding with zero-crossings in the imaginary part. It has been shown that the first occurrence of antiresonance in humans primarily reflects the contribution of the airways, and is a property of sound wave propagation in the airways. Also, the first antiresonance is altered in diseases such as chronic airflow obstruction, and wheeze in infants. The main aim of this project is to shed further understanding about the first antiresonance, via the behaviour of two parameters characterising this feature of the HF spectrum: the frequency at which the first antiresonance occurs, far,1, and the magnitude of the real part of Zrs at this frequency, Rrs(far,1). In our studies, Zrs is measured as an input impedance using the forced oscillation technique with a loudspeaker-in-box and wavetube setup, and employing pseudorandom frequency signals. We studied the effects of altered lung conditions, first in an animal model, then progressed on to humans. v vi In the rat, we found that both far,1 and Rrs(far,1) tended to decrease together with increasing lung volume. With methacholine(MCh)-induced bronchoconstriction, rats showed increases in far,1 and Rrs(far,1) with increasing MCh dose, but these occurred at higher doses compared to increases in airway resistance. The changes in these HF parameters were independent of the changes in tissue properties. ... It was found that in a group of patients with emphysema, VDRrs(far,1) was significantly more negative, potentially due to alterations to airway dimensions and wall properties. Furthermore, VDRrs(far,1) was correlated with extent of obstruction and hyperinflation, suggesting a relationship with severity of emphysema. These results show that the first antiresonance reflect changes in the airways, and its measurement shows promise as a clinical tool, in its potential as an easy-to-perform assessor of conditions in which the airways are altered. Keywords: antiresonance, respiratory input impedance, high frequencies, emphysema

Diss. (sammanfattning) Linköping : Univ., 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Com o intuito de avaliar sistema de suspensão mecânica da parede abdominal, elaborou-se protocolo experimental, estudando-se trinta cães, distribuídos em três grupos experimentais de maneira aleatória: grupo pneumoperitônio com gás carbônico, grupo suspensão pela cavidade peritoneal e grupo suspensão abdominal (intraperitoneal) pela tela subcutânea, para documentar alterações metabólicas, hemodinâmicas e respiratórias em cada grupo, compará-las e verificar possíveis vantagens ou desvantagens dos métodos. Os animais foram monitorizados com eletrocardiograma, cateter de Swan-Ganz e pressão arterial invasiva, sendo colhidas amostras de sangue arterial e venoso misto nos momentos basal, 0, 15, 30, 60, 90, 120 minutos e 30 minutos após o término do procedimento, anotando-se para cada momento os parâmetros hemodinâmicos. Calculouse a média e o desvio padrão dos parâmetros, sendo os diferentes grupos comparados pelo teste de Kruskal-Wallis, e nos momentos em que se encontrou diferença estatística entre os três grupos, comparou-se os grupos dois a dois pelo teste de Mann-Whitney. Observou-se, no grupo pneumoperitônio em relação aos grupos suspensão, queda significativa do pH sangüíneo, elevação do bicarbonato, elevação da pressão venosa central e elevação da pressão parcial de gás carbônico, diferenças estas estatisticamente significativas, com p<0,05, não havendo diferenças estatísticas entre os grupos suspensão abdominal e subcutânea / With the purpose of evaluate abdominal wall lifting system, a experimental protocol was elaborated, and thirty dogs, distributed randomly in three experimental groups, pneumoperitoneum with carbon dioxide gas, abdominal (intraperitoneal) suspension and subcutaneous suspension, were studied to document metabolic, hemodynamic and respiratory alterations, compare then and verify if there are some advantage or disadvantage in each method. The animals were monitored with electrocardiogram, Swan-Ganz catheter and invasive arterial blood pressure, and samples of arterial and venous blood were collected at the moments basal, 0, 15, 30, 60, 90 and 120 minutes, and 30 minutes after the end of procedure, registering at each moment the hemodynamical parameters. The three groups were statically compared with Kruskal-Wallis test and, at the moments with statistical differences, the groups were compared two by two with Mann-Whitney test. At the group pneumoperitoneum was observed significant decrease of blood pH, raising of bicarbonate, central venous pressure, and partial carbon dioxide blood pressure in relation to suspension groups, and those differences were statistically significant with p<0.05, without significant differences between abdominal and subcutaneous suspension groups